Therapeutic applications of estrogenic carboxylic acids

ABSTRACT

Provided are methods employing estrogenic compounds for: repressing weight gain or reducing weight in male patients; treating or preventing prostate cancer and peri- or post-menopausal symptoms; treating estrogen-responsive conditions that no longer respond to treatment with conventional steroidal estrogens; treating or preventing estrogen-responsive uterine cancer, breast cancer, and ovarian follicle atresia; inducing ovulation to increase fertility; oral contraception; treating or preventing diseases or conditions caused or prolonged by free radicals; treating or preventing cardiovascular disease, hyperlipidemia or hypercholesterolemia, and hyperglycemia; improving body fat distribution; and treating or preventing Alzheimer&#39;s disease, osteoporosis, and pattern baldness. Also provided are methods for treating or preventing prostatic diseases including benign prostate hyperplasia and other related conditions, androgen-responsive pathological conditions in males, and methods for male birth control and chemical castration, employing estrogenic carboxylic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser.No. 09/338,823, filed Jun. 23, 1999, which claims priority from U.S.Provisional Patent Application Serial No. 60/090,344, filed Jun. 23,1998.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of pharmaceuticaltherapeutics. More specifically, the present invention relates to theuse of estrogenic carboxylic acids in improved therapies for thetreatment of a variety of symptoms and disease conditions in mammals.The present invention also relates to the field of chemical synthesis,more specifically, the synthesis of estrogenic carboxylic acids.

BACKGROUND OF THE INVENTION

[0003] A. Estrogens

[0004] Estrogens, such as (+)-17β-estradiol (E2), have physiologicaleffects on males as well as females. In addition to their activity inreproductive tissue, they promote rapid weight gain in specific species,and have been marketed to fatten livestock quickly. Trenkle, AH: “TheMechanisms of Action of Estrogens in Feeds on Mammalian and AvianGrowth.” Proceedings of a Symposium: The Use of Drugs in Animal Feed.National Academy of Science, Washington D.C. 150-164 (1968); Meyers,U.S. Pat. No. 5,420,161. Estrogens have long been prescribed for theirbeneficial effects by reducing susceptibility to osteoporosis andameliorating menopausal and postmenopausal symptoms. Evans S F, Davie MW: “Low and Conventional Dose Transdermnal Oestradiol Are EquallyEffective at Preventing Bone Loss In Spine and Femur at AllPost-Menopausal Ages.” Clin Endocrinol. 44:79-84 (1996); Agarwal S K,Judd H L: “Menopause.” Curr Ther Endocrinol Metab. 6:624-631 (1997).Long-term clinical studies suggest that estrogens may be beneficial inpromoting cardiovascular health. Wilson P W: “The Impact of Estrogen onCardiovascular Disease.” Perspective Studies: The Framingham Study.Postgrad Med 51-53:89-90 (1989). More recently, estrogens have shownpromise as an adjunct in treatment of Alzheimer's disease. Filley C M:“AlZheimer's Disease in Women.” Am J Obstet Gynecol 176:1-7 (1997).Unfortunately, some estrogenic compounds administered in therapeuticdoses are suspected carcinogens in target tissues including breast anduterus. Persson I: “Cancer Risk in Women Receiving Estrogen-ProgestinReplacement Therapy.” Maturitas 23:S37-45 (1996).

[0005] Non-steroidal estrogens and antiestrogens, includingpharmaceuticals, environmental compounds, and phytochemicals, arecurrently receiving significant attention. This is understandable fromthe myriad potential applications increasingly being reported forestrogenic compounds, e.g., treating menopause- andpost-menopause-related problems, as anti-carcinogens, alleviatingosteoporosis, for contraceptive use, in estrogen-replacement therapy,treating prostatic disease, improving serum lipid profiles, etc. Themultiplicity of estrogenic effects now being discovered has led manyinvestigators to target specific populations for treatment with estrogenagonists and antagonists. Synthetic nonsteroidal compounds such astriphenylethylene derivatives (e.g., tamoxifen), dihydronapthalenederivatives (e.g., nafoxidine), and benzothiophene derivatives (e.g.,raloxifene) exhibit estrogenic and anti-estrogenic activity in varioustissues, these respective compounds showing specific advantages in themanagement of bone, uterine, serum cholesterol, and adipose tissue. See,generally, Trenkle, AH: “The Mechanisms of Action of Estrogens in Feedson Mammalian and Avian Growth.” Proceedings of a Symposium: The Use ofDrugs in Animal Feed. National Academy of Science, Washington D.C.150-164 (1968); Evans S F, Davie M W: “Low and Conventional DoseTransdermal Oestradiol Are Equally Effective at Preventing Bone Loss InSpine and Femur at All Post-Menopausal Ages.” Clin Endocrinol. 44:79-84(1996); Agarwal S K, Judd H L: “Menopause.” Curr Ther Endocrinol Metab.6:624-631 (1997); Wilson P W: “The Impact of Estrogen on CardiovascularDisease.” Perspective Studies:

[0006] The Framingham Study. Postgrad Med 51-53:89-90 (1989); Filley CM: “AlZheimer's Disease in Women.” Am J Obstet Gynecol 176:1-7 (1997);Persson I: “Cancer Risk in Women Receiving Estrogen-ProgestinReplacement Therapy.” Maturitas 23:S37-45 (1996); Heer J, Billeter J R,Miescher K: “Totalsynthese der racemischen bisdehydro-doisynolsäure.Über oestrogene carbosäuren IV.” Helv. Chim. Acta 28:1342-1354 (1945);Ke H Z, Chen H A, Simmons H A, Qi H, Crawford D T, Pirie C M,Chidsey-Frink K L, Ma Y F, Jee W S S, Thompson D D: “Comparative Effectsof Droloxifene, Tamoxifen, and Estrogen on Bone, Serum Cholesterol, andUterine Histology in the Ovariectomized Rat Model.” Bone 20:31-39(1997); Sato M, Rippy M K, Bryant H U: “Raloxifene, Tamoxifen,Nafoxidine, or Estrogen Effects on Reproductive and NonreproductiveTissues in Ovariectomized Rats.” FASEB J 10:905-912 (1996); Dodge J A,Glasebrook A L, Magee D A, Phillips D L, Sato M, Short L L, Bryant H U:“Environmental Estrogens: Effects on Cholesterol Lowering and Bone inthe Ovariectomized Rat.” J Steroid Biochem Molec Biol 59:155-161 (1996);Hart J E: “Endocrine Pathology of Estrogens: Species Differences.”Pharmac Ther 47:203-218 (1990); Heywood R, Wadsworth P F: “TheExperimental Toxicology of Estrogens.” Pharmac Ther 8:125-142 (1980);Baker V L, Draper M, Paul S, Allerheiligen S, Glant M, Shifren J, JaffeR B: “Reproductive Endocrine and Endometrial Effects of RaloxifeneHydrochloride, A Selective Estrogen Receptor Modulator, in Women withRegular Menstrual Cycles.” J Clin Endocrin Metab 83:6-13 (1998); DanZo BJ: “Environmental Xenobiotics May Disrupt Normal Endocrine Function byInterfering with the Binding of Physiological Ligands to SteroidReceptors and Binding Proteins.” Environ Health Perspect 105:294-301(1997); Baker V L, Jaffe R B: “Clinical Uses of Antiestrogens.” ObstetGynecol Surv 51:45-59 (1996); Knight D C, Eden J A: “A Review of theClinical Effects of Phytoestrogens.” Obstet Gynecol 87:897-904 (1996);Cooper R L, Kavlock R J: “Endocrine Disruptors and ReproductiveDevelopment: A Weight-of-Evidence Overview.” J Endocrinol 152:159-166(1997); Reubinoff B E, Wurtman J, Rojansky N, Adler D, Stein P, SchenkerJ G, BrZeZinski A: “Effects of Hormone Replacement Therapy on Weight,Body Composition, Fat Distribution, and Food Intake in EarlyPostmenopausal Women: A Prospective Study.” Fertil Steril 64:963-968(1995).

[0007] B. Doisynolic Acids and Related Estrogenic Compounds

[0008] Doisynolic acids, named after their discoverer, Edward Doisy, areestrogenic compounds originally obtained from alkali fusion of estroneand equilenin. “Doisynolic acid,” from estrone, contains a phenolicmoiety; and “bisdehydrodoisynolic acid” (BDDA), from equilenin,possesses a βnaphtholic moiety. Both types are seco-steroids, i.e., thesteroidal D-ring is cleaved. See Miescher K: “On Doisynolic Acids, A NewClass of Estrogens.” Chem Rev 43:367-384 (1948); Fieser L F, Fieser M:Natural Products Related to Phenanthrene, 347-353 (3rd Ed., ReinholdPublishing Corp., New York, N.Y. 1949). Meyers and Kolb reported theconversions of E2 and estrone under very mild conditions into doisynolicacids, which, in turn, exhibited estrogenic and antiestrogenic activitydepending on dosage. Meyers C Y, Kolb V M: “Facile and SelectiveChlorination and Cleavage of Some Cyclanones and Cyclanols With theCCl₄-KOH-t-BuOH Reagent. In situ Conversions of Estrones and Estradiolsinto Dichlorodoisynolic Acids.” J Org Chem 43:1985-1990 (1978). A numberof related pseudo-seco-steroid acids (most of them containing only tworings or a shifted C ring) also have been prepared. These compounds havebeen cited as exhibiting varying degrees of estrogenicity. Meyers C Y,Kolb V M, Gass G H, Rao B R, Roos C F, Dandliker W B: “Doisynolic-TypeAcids—Uterotropically Potent Estrogens which Compete Poorly withEstradiol for Cytosolic Estradiol Receptors. J Steroid Biochem31:393-404 (1988).

[0009] It has been reported that (±)-Z-doisynolic acid is moreestrogenic than (+)-E-doisynolic acid (C-14, S configuration) derivedfrom estrone or E2. Anner G, Miescher K: Hydrierungs-UndUmlagerungs-Reaktion in der Doisynolsäure—Reihe. Oestrogene CarbonsäurenXII. Helv. Chim. Acta 29 (1946) 1889-1895; and Die totalsyntheses vonracemischen doisynolsäuren XXI. Über oestrogene carbonsäueren. ibid30:1422-1432 (1947). Of the Z and E isomers of the doisynolic-typecompounds, (±)-Z-bisdehydrodoisynolic acid [(±)-Z-BDDA] has beenreported to be among the most estrogenic in vivo, rivaling or evensurpassing estradiol for vaginal cornification and uterotropism in thein vivo assays that were used to determine the comparativeestrogenicity. Miescher K: “On Doisynolic Acids, A New Class ofEstrogens.” Chem Rev 43:367-384 (1948); Fieser L F, Fieser M: NaturalProducts Related to Phenanthrene, 347-353 (3rd Ed., Reinhold PublishingCorp., New York, N.Y. 1949); Meyers C Y, Kolb V M, Gass G H, Rao B R,Roos C F, Dandliker W B: “Doisynolic-Type Acids—Uterotropically PotentEstrogens which Compete Poorly with Estradiol for Cytosolic EstradiolReceptors. J Steroid Biochem 31:393-404 (1988); Tschopp E: “Wirksamkeit,organconzentration und ausscheidung der7-methyl-bisdehydro-doisynolsäure.” Helv Physiol Pharmacol Acta4:401-410 (1946); Tschopp E: “Die oestrogene wirkung derbisdehydrodoisynolsäure und ihre derivate.” Helv Physiol Pharmacol Acta4:271-284 (1946); Rometsch R, Miescher K: “Die spaltung des racematesder n-bisdehydro-doisynolsäure. Über östrogene carbonsäuren X.” HelvChim Acta 29:1231-1235 (1946); and Terenius L: “Differential InhibitionIn Vitro of 17β-Estradiol Binding in the Mouse Uterus and Vagina byOptical Antipodes of Estrogen.” Molec Pharmac 4:301-310 (1968).Additional assays of (±)-Z-BDDA for estrogenicity, based on theestrogen-dependent cell proliferation in MCF-7 human mammary cancer cellline in culture, have confirmed the high estrogenic potency of thiscompound. Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos C F, Dandliker WB: “Doisynolic-Type Acids—Uterotropically Potent Estrogens which CompetePoorly with Estradiol for Cytosolic Estradiol Receptors. J SteroidBiochem 31:393-404 (1988); and Soto A M, Meyers C Y, Sonnenschein C:“How Many Rings Can be Cleaved from a Steroidal Estrogen WhilePreserving its Estrogenic Activity?” The Endocrine Society, 70th AnnualMeeting, Abstract (1988). And despite this estrogenic potency, the(±)-Z-BDDA has been reported to elicit neither toxicity norcarcinogenicity, even at 1000-times the estrogenic dosage. Meyers C Y,Kolb V M, Gass G H, Rao B R, Roos C F, Dandliker W B: “Doisynolic-TypeAcids—Uterotropically Potent Estrogens which Compete Poorly withEstradiol for Cytosolic Estradiol Receptors. J Steroid Biochem31:393-404 (1988). It has been reported that the (−) enantiomer ofZ-BDDA is the one responsible for the observed estrogenic potency. AnnerG, Miescher K: Hydrierungs—Und Umlagerungs-Reaktion in derDoisynolsäure—Reihe. Oestrogene Carbonsäuren XII. Helv. Chim. Acta 29(1946) 1889-1895; Die totalsyntheses von racemischen doisynolsäuren XXI.Über oestrogene carbonsäueren. ibid 30:1422-1432 (1947); Tschopp E:“Wirksamkeit, organconzentration und ausscheidung der7-methyl-bisdehydro-doisynolsäure.” Helv Physiol Pharmacol Acta4:401-410 (1946); Tschopp E: “Die oestrogene wirkung derbisdehydrodoisynolsäure und ihre derivate.” Helv Physiol Pharmacol Acta4:271-284 (1946); Rometsch R, Miescher K: “Die spaltung des racematesder n-bisdehydro-doisynolsäure. Über östrogene carbonsäuren X.” HelvChim Acta 29:1231-1235 (1946); and Terenius L: “Differential InhibitionIn Vitro of 17β-Estradiol Binding in the Mouse Uterus and Vagina byOptical Antipodes of Estrogen.” Molec Pharmac 4:301-310 (1968).

[0010] One of the distinctive properties of estrogenic doisynolic acidsis their very low binding affinity to cytosolic estrogen receptors whenconsidered in context with their very high in vivo activity. Thisanomaly was discovered by competitive binding inhibition studies with³H-estradiol using estrogen receptors extracted from rabbit uteri.Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos C F, Dandliker W B:“Doisynolic-Type Acids—Uterotropically Potent Estrogens which CompetePoorly with Estradiol for Cytosolic Estradiol Receptors. J SteroidBiochem 31:393-404 (1988). Unlabeled estradiol has been reported toinhibit this binding strongly, while the doisynoic acids have beenreported to do so only about 1% as well, despite being more active asestrogens in experimental animals. More recent direct binding studieswith ER α and ER β confirmed these results. Segaloff A.: “The Metabolismof Estrogens with Particular Emphasis on Clinical Aspects of Physiologyand Function of Ovarian Hormones.” Recent Progress in Hormone Research1949; IV:85-1 11; and Meyers C Y, Lutfi H G, Adler S: “TranscriptionalRegulation of Estrogen-Responsive Genes by Non-Steroidal Estrogens:Doisynolic and Allenolic acids.” J Steroid Biochem Molec Biol 62:477-489(1997).

[0011] Many recent studies have focused particularly on the in vivoactivity of (±)-Z-bisdehydrodoisynolic acid, the most active and easilyavailable doisynolic acid. Competitive binding-inhibition studies withuterine cytosolic estrogen receptors (ER) showed that the bindingaffinity of (±)-Z-BDDA was on the order of 0.01-0.03 of that of E2.Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos C F, Dandliker W B:“Doisynolic-Type Acids—Uterotropically Potent Estrogens which CompetePoorly with Estradiol for Cytosolic Estradiol Receptors. J SteroidBiochem 31:393-404 (1988); Soto A M, Meyers C Y, Sonnenschein C: “HowMany Rings Can be Cleaved from a Steroidal Estrogen While Preserving itsEstrogenic Activity?” The Endocrine Society, 70th Annual Meeting,Abstract (1988). Recent direct in vitro ER binding studies with human ERalpha (ER α) and ER beta (ER β) confirmed these results, in accord withthe binding affinities of (−)-Z-BDDA determined with mouse uterine ERpreparations in competitive binding-inhibition studies. Terenius L:“Differential Inhibition In Vitro of 17β-Estradiol Binding in the MouseUterus and Vagina by Optical Antipodes of Estrogen.” Molec Pharmac4:301-310 (1968); Segaloff A.: “The Metabolism of Estrogens withParticular Emphasis on Clinical Aspects of Physiology and Function ofOvarian Hormones.” Recent Progress in Hormone Research IV:85-111 (1949);and Meyers C Y, Lutfi H G, Adler S: “Transcriptional Regulation ofEstrogen-Responsive Genes by Non-Steroidal Estrogens: Doisynolic andAllenolic acids.” J Steroid Biochem Molec Biol 62:477-489 (1997). Unlikemost other estrogenic compounds studied with these techniques, the BDDAcompounds exhibit a low binding affinity accompanied by adisproportionately high biological activity. Without being bound by anyparticular theory, it is believed that the classic estrogen receptor,ER, may not be the exclusive receptor or pathway involved in mediatingthe actions of Z-BDDA and other estrogenic compounds; and/or thatdoisynolic acid compounds may act in vivo by some mechanism other thanby binding to estrogen cytosolic receptors to which estradiol, estrone,etc., normally bind. See Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos CF, Dandliker W B: “Doisynolic-Type Acids—Uterotropically PotentEstrogens which Compete Poorly with Estradiol for Cytosolic EstradiolReceptors. J Steroid Biochem 31:393-404 (1988).

[0012] Differences in the activity of E2 and (±)-Z-BDDA based on otherindices of estrogenic activity have also been observed. Specifically,while the rate of weight gain of female mice receiving E2 (e.g., 5g/animal/day) was increased over that of the control group, the rate ofweight gain of female mice receiving varying doses of (±)-Z-BDDA (e.g,5, 50, and 500 g/animal/day) was actually diminished. Meyers, U.S. Pat.No. 5,420,161.

[0013] While estradiol and its 3-methyl ether have been reported to beestrogenic in animals and humans, the 3-methyl ether of (±)-Z-BDDA hasonly been reported to be estrogenic in some animals (but inactive inhumans), and exhibits very little effect on proliferating human MCF-7cell growth. Soto A M, Meyers C Y, Sonnenschein C: “How Many Rings Canbe Cleaved from a Steroidal Estrogen While Preserving its EstrogenicActivity?” The Endocrine Society, 70th Annual Meeting, Abstract (1988).It has been hypothesized that the enzyme or receptor responsible for theconversion of the 3-methyl ether of estradiol to the estrogenic phenolicestradiol is present in animals (including humans), while that requiredfor the similar conversion of the 3-methyl ether of (±)-Z-BDDA ispresent in some animals, but not humans.

[0014] Despite the above-discussed advances, there still exists a needin the art for compounds exhibiting estrogen-like activity, but lackingthe undesirable side effects often observed in connection with the useof conventional estrogens, for use in methods for treating a widevariety of symptoms, conditions, and diseases responsive to estrogenscommonly employed at present.

[0015] C. Synthesis of Estrogenic Carboxylic Acids

[0016] In 1947 and 1948, Courrier, Horeau and Jacques (Courrier, R.;Horeau, A.; Jacques, J. Sur un nouvel oestrogene artificial de grandeactivité. Compt. rend. Soc. de biol. 1948, 141, 159-161; Horeau, A.;Jacques, J. Structure moleculaire et activité oestrogene: acideshydroxy-naphtylpropioniques substitutes. Acad. Sc. 1947, 224, 862-864;Courrier, R.; Horeau, A.; Jacques, J. L'acide allenolique et sesdérivés. Acad. Sc. 1947, 224,1401-1407; Courrier, R.; Horeau, A.;Jacques, J. Action de l'acide dimethyl-ethyl-allenolique chez la femellede cobaze qui allaite. Compt. rend. Soc. de biol. 1947, 141, 747;Jacques, J.; Horeau, A. Structure moleculaire et activité oestrogene(VI). Préparation de quelques dérivés de l'acide amphihydroxynaphtylβ-propionique (acide allenolique). Bull. Soc. Chim. France, 1948,711-716) reported the syntheses and biological studies of a series ofestrogenic compounds derived from 3-[2-(6-hydroxynaphthyl)]propionicacid 1, which was named allenolic acid in honor of Dr. E. Allen. Ofthese compounds, (−)-3-[2-(6-methoxynaphthyl)]-2,2-dimethyl-pentanoicacid 2 was found to exhibit the strongest estrogenic activity inanimals, including rats, cats, chicks, and guinea pigs, while the (+)enantiomer 3 showed only one-fifth the estrogenicity of 2 (Terenius, L.Inhibition of 17β-estradiol uptake on mouse uterus by doisynolic acidand allenolic acid derivatives: an in vitro differentiation between trueoestrogens and pro-oestrogens. Acta Pharmacol. et Toxicol., 1967, 25,313-322; Herbai, G. Separation of Growth Inhibition Potency fromOestrogenicity in Different Weak Oestrogenic Drugs of Various ChemicalStructures, Acta Endocrinologica, 1971, 68, 249-263). Later, the (−)enantiomer, 2, was marketed by G. D. Searle & Company under the tradename Vallestril® for the treatment of postmenopausal symptoms (Crawley,G. C. Hormones-nonsteroidal estrogens. In Kirk-Othmer Encyclo. Chem.Technol. 3rd Ed; Grayson, Martin, Eckroth, David, Eds; Wiley: New York,1980; vol. 12, 670-671).

[0017] Although 2 was highly estrogenic in animals, equivalent to17β-estradiol (E2), it was not found to have the same effects in womenas E2. In clinical trials, high dosages were required to elicit strongestrogenic responses from women (Sturnick, M. I.; Gargill, S. L.Clinical assay of a new synthetic estrogen: Vallestril. New England JMed., 1952, 247, 830-834; Schneeberg, N. G.; Perczek, L.; Nodine, J. H.;Perloff, W. H. Methallenstril, a new synthetic estrogen. J Am. Med.Assoc. 1956, 161, 1062-1067), and thus 2 was eventually removed from themarket.

[0018] In 1967, Terenius (Terenius, L. Inhibition of 17β-estradioluptake on mouse uterus by doisynolic acid and allenolic acidderivatives: an in vitro differentiation between true oestrogens andpro-oestrogens. Acta Pharmacol. et Toxicol., 1967, 25, 313-322) proposedthat 2 was a pro-estrogen, and that the true estrogen is its freephenolic form, i.e., compound 4, based on a study of the inhibition of17β-estradiol uptake in mouse uterus by those compounds. In 1971, Herbai(Herbai, G. Separation of Growth Inhibition Potency from Oestrogenicityin Different Weak Oestrogenic Drugs of Various Chemical Structures, ActaEndocrinologica, 1971, 68, 249-263) reported that in mice, compound 4exhibited a 100-fold stronger activity with regard to both inhibition ofweight gain and sulfate incorporation than compound 2. However, the (+)enantiomer of 4, compound 5, caused significant depression of sulfateincorporation without the corresponding effects on weight gain. Someyears later, Soto et al. (Soto, A. M.; Meyers, C. Y.; Sonnenschein, C.How Many Rings Can Be Cleaved from a Steroidal Estrogen while Preservingits Estrogenic Activity? The Endocrine Society, 70th Annual Meeting,Abstract (1988)) found that while 2 showed very little effect in humanMCF-7 cell proliferation, its phenolic form, 4, was found to be highlyeffective, suggesting that the low estrogenicity of 2 in women is due tohuman inability to cleave the methyl group from the ethereal oxygen.

[0019] Currently, there is a great deal of research interest inselective estrogen receptor modulators (SERMs) (Baker, V. L.; Draper,M.; Paul, S.; Allerheiligen, S.; Glant, M.; Shifren, J.; Jaffe, R. B.Reproductive endocrine and endometrial effects of raloxifenehydrochloride, a selective estrogen receptor modulator, in women withregular menstrual cycles. J. Clin. Endocrinol. Metab., 1998, 83, 6-13).SERMs have many potential medical applications, such as in treatingpostmenopausal symptoms, preventing osteoporosis, and hormonal therapyfor prostate cancer, while eliminating the unwanted side effects. Forexample, raloxifene is marketed by Eli Lilly under the trade nameEvista® to prevent osteoporosis in postmenpausal women while havinglittle effect on other reproductive organs. Recent studies on thephysiological effects of (+)- and (−)-cis-bisdehydrodoisynolic acids(cis-BDDA) in rats indicated that these compounds could be used in anumber of therapeutic applications (Banz, W. J.; Winters, T. A.; Hou,Y.; Adler, S.; Meyers, C. Y. Comparative Effects of (−)-, (+)- and(±)-Z-Bisdehydrodoisynolic Acids and Estradiol on Body Weight, FoodIntake and Metabolic Parameters in Male and Female Rats. Hormone andMetabolic Research, 1998, 30, 730-736). More importantly, (+)- and(−)-cis-BDDA have different physiological effects on various organs inintact rats. As estrogenic carboxylic acids, 4 and 5 have been shown tohave similar in vitro and in vivo biological properties to cis-BDDA(Meyers, C. Y.; Lutfi, H. G.; Adler, S. Transcriptional regulation ofestrogen-responsive genes by non-steroidal estrogens: Doisynolic andallenolic acids. J. Steroid Biochem. Molec. Biol., 62, 477-489 (1997)).

[0020] 3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid has anasymmetric center on the benzylic carbon. Thus, there exist twoenantiomers, as indicated by structures 2 and 3, above. Previoussyntheses all yielded a racemic mixture of 2 and 3. Thus, a resolutionprocess was required to obtain the desired enantiomer, as in the case ofVallestril®. Jacques and Horeau reported that quinine could be used toresolve the two enantiomers by forming two diastereomeric salts(Jacques, J.; Horeau, A. Structure moléculaire et activite oestrogène(VII). Dédoublement optique de l'acide α,α-diméthyl β-éthyl allenolique.iBull. Soc. Chim. France, 1949, 301-303).

[0021] Jacques and Horeau first reported the synthesis of(±)-3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid in 1947 andobtained a patent in 1949 (Scheme 1) (Jacques, J.; Horeau, A. Structuremoleculaire et activité oestrogene (VD). Préparation de quelques dérivésde l'acide amphihydroxynaphtyl β-propionique (acide allenolique). Bull.Soc. Chim. France, 1948, 711-716; Jacques, J.; Horeau, A. Naphthalenederivatives having estrogenic acitivity. Fr. Pat. 941,289 (1949)).

[0022] In 1948, Wieland and Miescher (Wieland, P.; Miescher, K.Estrogenic carboxylic acids. XXVI. Derivatives of alkylatedβ-naphthylvaleric acids. Helv. Chim. Acta, 1948, 31, 1844-1854) reporteda different synthesis of a racemic mixture of 2 and 3, and Gay andHoreau (Gay, R.; Horeau, A. Molecular structure and estrogenic activity.XV. Preparation of 2,2-dialkyl-3-(6-methoxy-2-naphthyl)pentanoic acidsand 2,2-dialkyl-3-(6-methoxy-2-naphthyl)hexanoic acids (derivatives ofallenolic acid). Bull. Soc. Chim. France, 1955, 955-962) alsosynthesized a racemic mixture through a similar route (Scheme 2). Thesesyntheses (Schemes 1 and 2) are multistep processes. After each step,separation of the intermediate product must be performed before it isused for the next reaction. Thus, additional chemicals, energy, andmanpower are needed, which increases the cost of production and lowersthe overall yield of the desired product.

[0023] Ciba Ltd. patented the shortest reported synthesis of racemicmixture of 2 and 3 so far in literature (Scheme 3) (Ciba Ltd,Naphthalenepropionic Acid, Swiss Patent 261,123 (1949); Ciba Ltd,Naphthalenepropionic Acids and Derivatives thereof, British Patent652,003 (1951)). Although there is only one step in this process, theyield of the product was not high due to self-coupling reactions.

[0024] All of these syntheses lead to a racemic mixture containing equalamounts of 2 and 3. Due to the different biological properties ofenantiomers 2 and 3, a resolution step must be performed to separate andisolate each enantiomer for pharmaceutical use, which also significantlyincreases the cost of production. In addition, the undesired enantiomer(50% of the racemic mixture) generated in the resolution process may bewasted if it is not used in other applications.

[0025] In the absence of a commercial source of 4 and 5, a one-pot,asymmetric synthesis of either 4 or 5 is needed in the art.

SUMMARY OF THE INVENTION

[0026] The present invention provides methods of using estrogeniccarboxylic acids and other non-steroidal estrogen-like compounds totreat or prevent a variety of conditions and diseases now being treatedwith conventional estrogens such as estradiol, ethinyl estradiol,estrone, or Premarin. The methods disclosed herein are based in part onthe emerging realization that the female hormones produced in males, andconversely male hormones produced in females, have far reaching effectsin health and disease, affording new approaches to a variety oftherapies. Further, the use of the estrogenic compounds disclosed hereinin the methods described below should result in improved therapieslacking the undesirable side effects often seen in connection with theuse of conventional estrogens.

[0027] Thus, in one aspect, the present invention provides a method forrepressing weight gain or reducing weight in a male patient, comprisingadministering (+)-Z-bisdehydrodoisynolic acid in a dosage effective torepress weight gain or reduce weight to a male patient suffering from,or disposed to, weight gain.

[0028] In another aspect, the present invention provides a method fortreating or preventing prostate cancer, comprising administering anestrogenic carboxylic acid in a dosage effective to treat or preventprostate cancer to a patient suffering from, or disposed to, prostatecancer. The estrogenic carboxylic acid can also be used to maintainprostate cancer patients who have been previously treated withinhibitors of gondadotropin releasing hormone (GnRH) secretion or oftestosterone. The predominant hormonal treatment now in use for prostatecancer consists of monthly injections of leuprolide, an antagonist ofGnRH. Hot flashes resulting from this treatment are a common complaint.In addition, leuprolide, a polypeptide, may give rise to an immuneresponse on continued use. In contrast, the estrogenic carboxylic acidsof the present invention are almost certainly non-immunogenic. Thesecompounds should reduce the size of the testes, thereby ameliorating theeffects of prostate hyperplasia, limiting the spread of prostate cancercells.

[0029] In another aspect, the present invention provides a method fortreating or preventing peri- or post-menopausal symptoms, comprisingadministering an estrogenic carboxylic acid in a dosage effective totreat or prevent peri- or post-menopausal symptoms to a patientsuffering from, or disposed to, said menopausal symptoms. The presentestrogenic carboxylic acids can be used in place of conventionalestrogens in hormone replacement therapy in menopause.

[0030] In another aspect, the present invention provides a method fortreating an estrogen-responsive condition that no longer responds totreatment with conventional steroidal estrogens, comprisingadministering an estrogenic carboxylic acid in a dosage effective torepress, reduce, or otherwise ameliorate said condition to a patientsuffering from said condition.

[0031] In yet another aspect, the present invention provides a methodfor treating or preventing an estrogen-responsive uterine cancer,comprising administering an estrogenic carboxylic acid in a dosageeffective to treat or prevent said cancer to a patient suffering from,or disposed to, said cancer.

[0032] In yet another aspect, the present invention provides a methodfor treating or preventing breast cancer, comprising administering anestrogenic carboxylic acid in a dosage effective to treat or preventsaid cancer to a patient suffering from, or disposed to, breast cancer.

[0033] These methods of treating uterine cancer and breast cancer arebased on the estrogenic, antiestrogenic, and antioxidant properties ofthe present estrogenic carboxylic acids.

[0034] In another aspect, the present invention provides a method fortreating or preventing ovarian follicle atresia, comprisingadministering an estrogenic carboxylic acid in a dosage effective totreat or prevent ovarian follicle atresia to a patient suffering from,or disposed to, atresia.

[0035] In a further aspect, the present invention provides a method forinducing ovulation to increase fertility, comprising administering anestrogenic carboxylic acid in a dosage effective to induce ovulation toa patient suffering from, or disposed to, ovulatory disorder. Theestrogenic carboxylic acid can be administered during the mid-portion ofthe first part of the menstrual cycle, for example, for five days,starting at the fifth day of said menstrual cycle.

[0036] In yet a further aspect, the present invention provides a methodfor oral contraception, comprising administering an estrogeniccarboxylic acid in a dosage effective to prevent ovulation to saidpatient throughout the menstrual cycle, starting at day one thereof andcontinuing throughout said menstrual cycle to about day 21. This methodis especially useful for treatment of a patient not suitable fortreatment with a steroidal estrogen, for example one who is a tobaccosmoker, an obese patient, a patient suffering from breast disease, or apatient prone to producing emboli. In obese patients, this methodprovides the added benefit of promoting concomitant weight loss. Inthese methods, the estrogenic carboxylic acid can be administered incombination with a progestin.

[0037] In another aspect, the present invention provides a method fortreating or preventing a disease or condition caused or prolonged byfree radicals, comprising administering an estrogenic carboxylic acid ina dosage effective to treat or prevent said disease or condition to apatient suffering from, or disposed to, said disease or condition.

[0038] Another aspect of the present invention provides a method fortreating or preventing cardiovascular disease, comprising administeringan estrogenic carboxylic acid in a dosage effective to treat or preventcardiovascular disease to a patient suffering from, or disposed to,cardiovascular disease.

[0039] In another aspect, the present invention provides a method fortreating or preventing hyperlipidemia or hypercholesterolemia,comprising administering an estrogenic carboxylic acid in a dosageeffective to treat or prevent hyperlipidemia or hypercholesterolemia toa patient suffering from, or disposed to, hyperlipidemia orhypercholesterolemia.

[0040] In another aspect, the present invention provides a method fortreating or preventing hyperglycemia, comprising administering anestrogenic carboxylic acid in a dosage effective to treat or preventhyperglycemia to a patient suffering from, or disposed to,hyperglycemia.

[0041] Yet another aspect of the present invention involves a method forimproving body fat distribution, comprising administering an estrogeniccarboxylic acid in a dosage effective to improve body fat distributionto a patient suffering from, or disposed to, abnormal body fatdistribution.

[0042] A further aspect of the present invention relates to a method fortreating or preventing Alzheimer's disease, comprising administering anestrogenic carboxylic acid in a dosage effective to treat or preventAlzheimer's disease to a patient suffering from, or disposed to,Alzheimer's disease.

[0043] Yet a further aspect of the present invention relates to a methodfor treating or preventing osteoporosis, comprising administering anestrogenic carboxylic acid in a dosage effective to treat or preventosteoporosis to a patient suffering from, or disposed to, osteoporosis.

[0044] In still another aspect, the present invention provides a methodfor treating or preventing pattern baldness, comprising administering anestrogenic carboxylic acid in a dosage effective to treat or preventpattern baldness to a patient suffering from, or disposed to, patternbaldness. Such patients include both males and females. In balding men,hair growth should be stimulated by the reduction of testosterone levelsproduced by feedback inhibition of the pituitary occasioned by the risein estrogen.

[0045] In another aspect, the present invention provides a method fortreating or preventing a prostatic disease or condition, comprisingadministering an estrogenic carboxylic acid in a dosage effective totreat or prevent a prostatic disease or condition to a patient sufferingfrom, or disposed to, such disease or condition. (+)-Z-BDDA, (−)-Z-BDDA,(−)-HAA, (+)-HAA, can be used in this method, with (+)-Z-BDDA beingpreferred. Examples of prostatic diseases and conditions amenable tosuch treatment include, but are not limited to, prostate cancer, benignprostate hypertrophy, and prostatitis. These and other prostaticdiseases and conditions can be treated without negative side effectssuch as testis shrinkage, inhibition of spermatogenesis, gynecomastia,or other feminizing effects in males in accordance with this method.

[0046] In another aspect, the present invention provides a method fortreating or preventing an androgen-responsive pathological condition ina male, comprising administering an estrogenic carboxylic acid in adosage effective to treat or prevent said pathological condition to amale patient suffering from, or disposed to, said pathologicalcondition.

[0047] In yet another aspect, the present invention provides a method ofbirth control, comprising administering an estrogenic carboxylic acid ina dosage effective to inhibit spermatogenesis in a male. Compoundsuseful in this method include, but are not limited to, (−)-Z-BDDA,(−)-HAA, and (+)-HAA.

[0048] In a still further aspect, the present invention provides amethod for chemical castration in a male, comprising administering anestrogenic carboxylic acid in a dosage effective to shrink the testis orcause a loss of libido and/or impotence in a male. Compounds useful inthis method include, but are not limited to, (−)-Z-BDDA, (−)-HAA, and(+)-HAA.

[0049] Treatment of the foregoing symptoms, conditions, and diseaseswith the compounds of the present invention should be accompanied byfewer side effects than are often observed in connection with the use ofconventional estrogens.

[0050] In any of the foregoing methods, the estrogenic therapeuticcompound most preferably is an estrogenic carboxylic acid, such as, forexample, a doisynolic acid, an allenolic acid, aphenylcyclohexenecarboxylic acid, a hydroxyphenylcyclo-hexenecarboxylicacid, a phenylcyclohexanecarboxylic acid, ahydroxyphenylcyclohexanecarboxylic acid, ahydroxytetrahydro-anthracenecarboxylic acid, or atetrahyroanthracene-carboxylic acid. More specifically, the estrogeniccarboxylic acid can be, for example, (+)-doisynolic acid,(−)-Z-bisdehydrodoisynolic acid, (+)-Z-bisdehydrodoisynolic acid,(±)-Z-bisdehydro-doisynolic acid (Z-BDDA), (−)-allenolic acid,(+)-allenolic acid,1-(p-hydroxyphenyl)-6-ethyl-5-methylcyclohexene-4-carboxylic acid,1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohexene-4-carboxylic acid,1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohexene-4-carboxylicacid, 4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexanecarboxylic acid,1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2-carboxylicacid, 1-phen yl-2-ethyl-3-methylcyclohexene-4-carboxylic acid, and1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid. Derivatives of suchcompounds (e.g., a pharmaceutically acceptable salt, ester, oranhydride) may also be used. In the methods disclosed herein, theseestrogenic carboxylic acids can be used alone or in combination.

[0051] In yet another aspect, the present invention provides a directone-pot synthesis to produce esters of3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid from commerciallyavailable starting material. These esters can then be easily hydrolyzedunder basic or acidic conditions to yield the corresponding acids 2 or3, discussed above.

[0052] Further scope of the applicability of the present invention willbecome apparent from the detailed description and drawings providedbelow. However, it should be understood that the following detaileddescription and examples, while indicating preferred embodiments of theinvention, are given by way of illustration only since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above and other objects, features, and advantages of thepresent invention will be better understood from the following detaileddescription taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

[0054]FIG. 1 shows results of Experiment 2 of Example 2, i.e., theeffects of(−)-, (+)-, and (±)-Z-bisdehydrodoisynolic acid (Z-BDDA) and(+)-17βb-estradiol on uterus weight in rats on treatment for 5-6 weeks[¹significantly different from vehicle (p<0.05) and ²significantlydifferent from (−)-Z-BDDA (p<0.05), n=5/treatment, all values are themean±SEM].

[0055]FIG. 2 shows results of Experiment 1 of Example 2, i.e., theeffects of (+)- and (±)-Z-bisdehydrodoisynolic acid (Z-BDDA) and(+)-17βestradiol on percent weight change in male and female rats ontreatment for 4 weeks [¹significantly different from control (p<0.05);²significantly different from vehicle (p<0.05); ³ significantlydifferent from estradiol (p<0.05); and ⁴Significantly different from(±)-Z-BDDA (p<0.05), n=5/treatment. All values are the mean ±SEM].

[0056]FIG. 3 shows results of Experiment 2 of Example 2, i.e., theeffects of(−)-, (+)-, and (±)-Z-bisdehydrodoisynolic acid (Z-BDDA) and(+)-17βestradiol on % weight change in male and female rats on treatmentfor 5-6 weeks [¹significantly different from vehicle (p<0.05) and²significantly different from estradiol (p<0.05), n=5/treatment, allvalues are the mean±SEM].

[0057] FIGS. 4a-4f show the effects of (−)- and(+)-Z-bisdehydrodoisynolic acids (BDDA), (−)- and (+)-hydroxyallenolicacid (HAA), and (+)-17β-estradiol (E) on prostate histology in male ratson treatment for 6 weeks (Example 4). Photomicrographs representhemotoxylin and eosin stains of paraffin sections photographed at 20×.Representative panels were treated as labeled at 0.1 μg/g bodyweight.

[0058] FIGS. 5a-5f show the effects of (−)- and(+)-Z-bisdehydrodoisynolic acids (BDDA), (−)- and (+)-hydroxyallenolicacid (HAA), and (+)-17β-estradiol (E) on testis histology in male ratson treatment for 6 weeks (Example 4). Photomicrographs representhemotoxylin and eosin stains of paraffin sections photographed at 20×.Representative panels were treated as labeled at 0.1 μg/g bodyweight.

[0059]FIG. 6 shows the results of the lag time oxidation studiesdescribed in Example 5. Compounds shown on the x-axis, from left toright: daidzein, genistein, 4-hydroxy-tamoxifen, (+)-allenolic acid,(−)-allenolic acid, (+)-Z-bisdehydrodoisynolic acid,(−)-Z-bisdehydrodoisynolic acid. Each compound was tested at aconcentration of 10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁷ M. The results obtained ateach of these concentrations is shown for each compound, from left toright as a vertical bar, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0060] Although the following detailed description is provided to aidthose skilled in the art in practicing the present invention, it shouldnot be construed to unduly limit the present invention. Modificationsand variations in the embodiments discussed herein can be made by thoseof ordinary skill in the art without departing from the spirit or scopeof the present inventive discovery.

[0061] The present methods utilize the unique properties of doisynolicacid and related estrogenic compounds (especially related estrogeniccarboxylic acids) in animal (particularly human) therapy and research.Based on the properties of these compounds, together with presentmethods of treating prostate, breast, and uterine cancer, osteoporosis,atresia, Alzheimer's disease, etc., improved therapies are proposed forthese disorders. These improvements stem from the unique properties ofthe estrogenic therapeutic compounds discussed below, which place themin a separate category of estrogenic compounds distinguished from theconventional physiological estrogens (e.g., estradiol and estrone).These properties include low toxicity, long acting effect, and theabsence of any detectable carcinogenicity.

[0062] The estrogenic therapeutic compounds (particularly estrogeniccarboxylic acids) used in accordance with this invention may also be avaluable research tool for testing the estrogen receptors and/orpathways involved in mediating the actions elicited by estrogenic andnon-estrogenic compounds. As noted earlier, there is an apparentactivity/binding paradox suggesting that the classic estrogen receptor,ER α, may not be the exclusive receptor or pathway mediating the actionsof Z-BDDA compounds, or possibly even those of estradiol. Initialstudies comparing the classical ER α and the novel estrogen receptor ERβ show similar results. The binding affinity of (+)-Z-BDDA is even lowerthan that of the (−)-enantiomer, and both enantiomers have a much loweraffinity for estrogen receptors than does estradiol, where measured viadirect receptor binding assays or by generating does-response profilesusing activation of estrogen-responsive reportes genes in cell-culturesystems. Indeed, estradiol and other conventional estrogenic compoundsappear to be dependent on high affinity binding to ER α and/or β foreliciting the classical estrogen response. This effect does not appearto be the case for the present estrogenic therapeutic compounds(particularly the estrogenic carboxylic acids), which suggests thatthese compounds are selective estrogen response modulators (SERMs) thatelicit their estrogenic actions by a novel binding of the estrogenreceptors, or by a mechanism that is independent of the estrogenreceptor and which occurs elsewhere in the estrogen response pathway.Therefore, these estrogenic therapeutic compounds (particularlyestrogenic carboxylic acids) have the potential for use as researchtools in determining if a classical or novel estrogenic action isdependent on high affinity binding to ER α and/or β, or is elicited vialow affinity binding to the estrogen receptors. Additionally, thepresent estrogenic compounds can be a valuable tool in elucidating andcharacterizing the mechanisms involved in the classical and novelestrogen signaling pathway, testing the estrogen receptors and/orpathways involved in mediating the actions elicited by estrogenic andnon-estrogenic compounds. Sites of action of the present estrogeniccompounds can also be detected by transcriptional initiation through acotransfection assay.

[0063] A. Compounds

[0064] The estrogenic compounds useful in the methods of the presentinvention include, for example, doisynolic acid compounds,bisdehydrodoisynolic acid compounds, allenolic acid compounds, phenyl-and hydroxyphenylcyclohexane compounds, phenyl- andhydroxyphenyl-cyclohexene compounds, and hydroxytetrahydroanthracenecompounds. Especially preferred compounds include, for example,doisynolic acid, bisdehydrodoisynolic acid, allenolic acid, phenyl- andhydroxyphenylcyclohexane carboxylic acids, phenyl- andhydroxyphenyl-cyclohexene carboxylic acids, andhydroxytetrahydroanthracene carboxylic acids.

[0065] In addition to the foregoing specific compounds, a number ofderivatives thereof are also contemplated for use in the presentmethods. These include hydroxynaphthyl alkylated alkanoic acids,hydroxyphenyl alkylated cyclohexene- and cyclohexanecarboxylic acids,and hydroxyalkylated tetrahydro-anthracenecarboxylic acids, and thecorresponding non-hydroxylated compounds which are hydroxylated in vivo,and thereby likewise exhibit estrogenic activity. Methods for preparingthese derivatives involve conventional synthetic organic chemicalreactions within the ordinary skill of the art.

[0066] In the various therapeutic methods disclosed herein, one can usethe estrogenically active compounds of the present invention in theirphenolic or non-phenolic forms, as free acids, or correspondingpharmaceutically acceptable salts, ethers, esters, or other easilyhydrolyzable derivatives such as anhydrides, etc.

[0067] The preferred structures of the compounds for use in accordancewith this invention are shown below. It should be noted that in somecases, only one enantiomeric structure is illustrated for each of aparticular compound. However, as these compounds possess asymmetriccarbon atoms, enantiomers and diastereomers other than those shown mayexhibit specific biological activity. As it is known to those skilled inthe art that the compounds of the present invention having suchasymmetric carbon atoms can exist in enantiomeric, diastereomeric, andracemic forms, all these forms are contemplated within the scope of thepresent invention. More specifically, the present invention includessuch enantiomers, diastereomers, racemic mixtures, and other mixturesthereof.

[0068] In one of the more preferred embodiments of this invention, thetherapeutic estrogenic compound has the structure of formula (I) or is apharmaceutically acceptable salt thereof:

[0069] Here, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, and X¹⁰ preferably arecarbon atoms.

[0070] The dashed lines are optional π bonds (i.e., a bond representedby both a solid line and a dashed line may optionally be either a singleor a double bond).

[0071] R^(1β), R^(2β), R^(3β), R^(4β), R⁵, R^(6β), R^(7β), R^(8β),R^(9β), and/or R¹⁰ are present only when X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸,X⁹, and/or X¹⁰, respectively, are saturated (i.e., are bound to 4atoms).

[0072] R^(1α) and R^(2α) preferably are independently a moiety which (a)comprises from 1 to 20 carbon atoms (more preferably from 1 to 6 carbonatoms), and is selected from the group consisting of hydrocarbyl andsubstituted hydrocarbyl (i.e., R^(1α) and R^(2α) are independently acarbon-containing moiety which comprises no greater than 20 carbonatoms, and more preferably no greater than 6 carbon atoms); or (b) doesnot comprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, imino, nitro, nitroso, oximido, oxo, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl,silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo.More preferably, R^(1α) and R^(2α) are independently selected from thegroup consisting of hydrogen; hydrocarbyl comprising from 1 to 6 carbonatoms; and —OR¹⁰⁰, wherein R¹⁰⁰ is hydrogen or hydrocarbyl containingfrom 1 to 6 carbon atoms, and particularly wherein R¹⁰⁰ is hydrogen ormethyl. Most preferably, R^(1α) and R^(2α) are hydrogen.

[0073] R^(1β), R^(2β), R^(3β), R^(4β), R⁵, R^(6β), R^(8β), R^(9β), andR¹⁰ preferably are independently a moity which (a) comprises from 1 to20 carbon atoms (more preferably from 1 to 6 carbon atoms), and isselected from the group consisting of hydrocarbyl and substitutedhydrocarbyl; or (b) does not comprise a carbon atom and is selected fromthe group consisting of amino, halogen, hydrogen, nitro, nitroso, oxy,phosphino, phosphinyl, phospho, phosphono, phosphoranyl, phosphoroso,siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and thio. Morepreferably, R^(1β), R^(2β), R^(3β), R^(4β), R⁵, R^(6β), R^(8β), R^(9β),and R¹⁰ are independently selected from the group consisting of hydrogenand hydrocarbyl comprising from 1 to 6 carbon atoms. More preferably,R^(1β), R^(2β), R^(3β), R^(4β), R⁵, R^(6β), R^(8β), R^(9β), and R¹⁰ arehydrogen.

[0074] R^(3α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, imino, nitro, nitroso, oximido, oxo, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl,silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo.In a more preferred embodiment, R^(3α) is hydrogen. In another morepreferred embodiment, R^(3α) is selected from the group consisting ofglycosidyl, acetylated glycosidyl, and malonylated glycosidyl. In anadditional more preferred embodiment, R^(3α) is —OC(O)(R¹⁰¹), whereinR¹⁰¹ is benzyl or —N(CH₂CH₂Cl)₂. In yet another more preferredembodiment, R^(3α) comprises (a) no greater than 20 carbon atoms (morepreferably, no greater than 6 carbon atoms); and (b) a moiety selectedfrom the group consisting of amino, halogen, hydrogen, imino, oximido,oxo, oxy, phosphinidene, phosphino, phosphinyl, phosphinylidene,phosphono, phosphoranyl, phosphoranylidene, siloxy, silyl, silylene,sulfeno, sulfino, sulfo, and thio. In an even more preferred embodiment,R^(3α) comprises (a) no greater than 20 carbon atoms (more preferably,no greater than 6 carbon atoms); and (b) a moiety selected from thegroup consisting of amino, imino, oximido, oxy, phosphinidene,phosphino, phosphinyl, phosphinylidene, phosphono, phosphoranyl,phosphoranylidene, siloxy, silyl, silylene, sulfeno, sulfino, sulfo, andthio. In a still even more preferred embodiment, R^(3α) comprises apolarizable hydrogen atom (i.e., a hydrogen atom bound to an atom otherthan a carbon atom), and is, for example, —C(O)(OH), —NH₂, ═NH, ═N(OH),—OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H), —P(O)(OH)(OH), —PH₄, ═PH₃, ═SiH₂,—S(OH), —S(O)(OH), S(O)(O)(OH), or —SH. In an alternative even morepreferred embodiment, R^(3α) is —OR¹⁰² or —OC(O)(R¹⁰³), wherein R¹⁰² andR¹⁰³ are hydrogen, halogen, or hydrocarbyl comprising from 1 to 19carbon atoms (particularly 1 to 5 carbon atoms, and more particularlyfrom 1 to 2 carbon atoms). Most preferably, R^(3α) is —OH.

[0075] R^(4α), R^(6α), and R^(9α) preferably are independently a moietywhich (a) comprises from 1 to 20 carbon atoms (more preferably from 1 to6 carbon atoms), and is selected from the group consisting ofhydrocarbyl and substituted hydrocarbyl; or (b) does not comprise acarbon atom and is selected from the group consisting of amino, halogen,hydrogen, imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene,phosphino, phosphinyl, phosphinylidene, phospho, phosphono,phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl, silylene,sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. Morepreferably, R^(4α), R^(6α), and R^(9α) are independently selected fromthe group consisting of hydrogen and hydrocarbyl comprising from 1 to 6carbon atoms. Most preferably, R^(4α), R^(6α), and R^(9α) are hydrogen.

[0076] R^(7α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, imino, nitro, nitroso, oximido, oxo, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl,silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo.More preferably, R^(7α) is selected from the group consisting ofhydrogen, hydrocarbyl comprising from 1 to 20 carbon atoms, and oxo.Most preferably, R^(7α) is hydrogen.

[0077] R^(7β) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. More preferably, R^(7β) is selectedfrom the group consisting of hydrogen and hydrocarbyl comprising from 1to 6 carbon atoms. Most preferably, R^(7β) is hydrogen.

[0078] R^(8α) preferably is a substituted hydrocarbyl comprising amoiety selected from the group consisting of amino, imino, oximido, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phosphono,phosphoranyl, phosphoranylidene, siloxy, silyl, silylene, sulfeno,sulfino, sulfo, and thio. More preferably, R^(8α) comprises asubstituted hydrocarbyl containing a polarizable hydrogen atom (R^(8α)is, for example, —C(O)(OH), —NH₂, ═NH, ═N(OH), —OH, ═PH, —PH₂,—P(O)(H)(H), ═P(O)(H), —P(O)(OH)(OH), —PH₄, ═PH₃, ═SiH₂, —S(OH),—S(O)(OH), S(O)(O)(OH), or —SH). In an alternative more preferredembodiment, R^(8α) is a substituted hydrocarbyl comprising —C(O)(OR¹⁰⁴),wherein R¹⁰⁴ is hydrogen, halogen, or hydrocarbyl comprising from 1 to 5carbon atoms (particularly from 1 to 2 carbon atoms). Most preferably,R^(8α) is a substituted hydrocarbyl comprising —C(O)(OH), making thecompound an estrogenic carboxylic acid. In a particularly preferredembodiment, R^(8α) comprises no greater than 20 carbon atoms.

[0079] In some instances, it is preferred that the therapeutic compoundof formula (I) have the structure of formula (II) or be apharmaceutically acceptable salt thereof:

[0080] Here, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, R^(1α), R^(1β),R^(2α), R^(2β), R^(3α), R^(3β), R^(4α), R^(4β), R⁵, R^(6α), R^(6β),R^(7α), R^(7β), R^(8β), R^(9α), R^(9β), and R¹⁰ are preferably asdefined above for formula (I).

[0081] X¹³ and X¹⁴ preferably are carbon atoms.

[0082] The dashed lines are optional π bonds.

[0083] R^(1β), R^(2β), R^(3β), R^(4β), R⁵, R^(6β), R^(7β), R^(8β),R^(9β), R¹⁰, R^(13γ), and/or R^(14β) are present only when X¹, X², X³,X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹³, and/or X¹⁴, respectively, aresaturated.

[0084] R^(13α) preferably comprises (a) no greater than 20 carbon atoms(more preferably no greater than 6 carbon atoms); and (b) a moietyselected from the group consisting of amino, imino, oximido, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phosphono,phosphoranyl, phosphoranylidene, siloxy, silyl, silylene, sulfeno,sulfino, sulfo, and thio. More preferably, R^(13α) comprises apolarizable hydrogen atom and is, for example, —C(O)(OH), —NH₂, ═NH,═N(OH), —OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H), —P(O)(OH)(OH), —PH₄,═PH₃, ═SiH₂, —S(OH), —S(O)(OH), S(O)(O)(OH), or —SH. In an alternativemore preferred embodiment, R^(13α) is —C(O)(OR¹⁰⁵), wherein R¹⁰⁵ ishydrogen, halogen, or hydrocarbyl comprising from 1 to 5 carbon atoms.Most preferably, R^(13α) is —C(O)(OH) (i.e., the compound is anestrogenic carboxylic acid).

[0085] R^(13β) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably no greater than 6 carbon atoms), and is selected from thegroup consisting of hydrocarbyl and substituted hydrocarbyl; or (b) doesnot comprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, imino, nitro, nitroso, oximido, oxo, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl,silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo.More preferably, R^(13β) is selected from the group consisting ofhydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms. Even morepreferably, R^(13β) is are independently hydrocarbyl comprising from 1to 6 carbon atoms. Most preferably, R^(13β) is methyl.

[0086] R^(13γ) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably no greater than 6 carbon atoms), and is selected from thegroup consisting of hydrocarbyl and substituted hydrocarbyl; or (b) doesnot comprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. More preferably, R^(13γ) is selectedfrom the group consisting of hydrogen and hydrocarbyl comprising from 1to 6 carbon atoms. Even more preferably, R^(13γ) is hydrocarbylcomprising from 1 to 6 carbon atoms. Most preferably, R^(13γ) is methyl.

[0087] R^(14α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably no greater than 6 carbon atoms), and is selected from thegroup consisting of hydrocarbyl and substituted hydrocarbyl; or (b) doesnot comprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, imino, nitro, nitroso, oximido, oxo, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl,silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo.More preferably, R^(14α) is selected from the group consisting ofhydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms. Even morepreferably, R^(14α) is hydrocarbyl comprising from 1 to 6 carbon atoms.Most preferably, R^(14α) is ethyl.

[0088] R^(14β) preferably (a) comprises from 1 to 20 (more preferably nogreater than 6 carbon atoms), carbon atoms and is selected from thegroup consisting of hydrocarbyl and substituted hydrocarbyl; or (b) doesnot comprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. More preferably, R^(14β) is selectedfrom the group consisting of hydrogen and hydrocarbyl comprising from 1to 6 carbon atoms. Most preferably, R^(14β) is hydrogen.

[0089] One particularly preferred structure of formula (II) is“allenolic acid” (or “AA”), which has the following formula (M):

[0090] In other instances, it is particularly preferred for R^(8α) toform a carbocyclic ring with R^(9α) in formula (I) to create, forexample, a doisynolic acid derivative having the formula (IV) or apharmaceutically acceptable salt thereof:

[0091] Here, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, R^(1α), R^(1β),R^(2α), R^(2β), R^(3α), R^(3β), R^(4α), R^(4β), R⁵, R^(6α), R^(6β),R^(7α), R^(7β), R^(8β), R^(9β), and R¹⁰ are preferably as defined abovefor formula (I).

[0092] X¹¹, X¹², X¹³ and X¹⁴ preferably are carbon atoms.

[0093] The dashed lines are optional π bonds.

[0094] R^(1β), R^(2β), R^(3β), R^(4β), R⁵, R^(6β), R^(7β), R^(8β),R^(9β), R¹⁰, R^(11β), R^(12β), R^(13β), and/or R^(14β) are present onlywhen X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹³, and/or X¹⁴,respectively, are saturated.

[0095] R^(11α) and R^(12α) preferably are independently a moiety which(a) comprises from 1 to 20 carbon atoms (more preferably, from 1 to 6carbon atoms), and is selected from the group consisting of hydrocarbyland substituted hydrocarbyl; or (b) does not comprise a carbon atom andis selected from the group consisting of amino, halogen, hydrogen,imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More preferably,R^(11α) and R^(12α) are independently selected from the group consistingof hydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms. Mostpreferably, R^(11α) and R^(12α) are hydrogen.

[0096] R^(11β), R^(12β), and R^(14β) preferably are independently amoiety which (a) comprises from 1 to 20 carbon atoms (more preferably,from 1 to 6 carbon atoms), and is selected from the group consisting ofhydrocarbyl and substituted hydrocarbyl; or (b) does not comprise acarbon atom and is selected from the group consisting of amino, halogen,hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl, phospho,phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino,sulfo, sulfonyl, and thio. More preferably, R^(11β), R^(12β), andR^(14β) are independently selected from the group consisting of hydrogenand hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,R^(11β), R^(12β), and R^(14β) are hydrogen.

[0097] R^(13α) preferably comprises (a) no greater than 20 carbon atoms(more preferably no greater than 6 carbon atoms); and (b) a moietyselected from the group consisting of amino, imino, oximido, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phosphono,phosphoranyl, phosphoranylidene, siloxy, silyl, silylene, sulfeno,sulfino, sulfo, and thio. More preferably, R^(13α) comprises apolarizable hydrogen atom and is, for example, —C(O)(OH), —NH₂, NH,═N(OH), —OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H), —P(O)(OH)(OH), —PH₄,═PH₃, ═SiH₂, —S(OH), —S(O)(OH), S(O)(O)(OH), or —SH. In an alternativemore preferred embodiment, R^(13α) is —C(O)(OR¹⁰⁶), wherein R¹⁰⁶ ishydrogen, halogen, or hydrocarbyl comprising from 1 to 5 carbon atoms.Most preferably, R^(13α) is —C(O)(OH) (i.e., the compound is anestrogenic carboxylic acid).

[0098] R^(13β) preferably (a) comprises from 1 to 20 carbon atoms and isselected from the group consisting of hydrocarbyl and substitutedhydrocarbyl; or (b) does not comprise a carbon atom and is selected fromthe group consisting of amino, halogen, hydrogen, nitro, nitroso, oxy,phosphino, phosphinyl, phospho, phosphono, phosphoranyl, phosphoroso,siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and thio. Morepreferably, R^(13β) is selected from the group consisting of hydrogenand hydrocarbyl comprising from 1 to 6 carbon atoms. Even morepreferably, R^(13β) is hydrocarbyl comprising from 1 to 6 carbon atoms.Most preferably, R^(13β) is methyl.

[0099] R^(14α) preferably (a) comprises from 1 to 20 carbon atoms and isselected from the group consisting of hydrocarbyl and substitutedhydrocarbyl; or (b) does not comprise a carbon atom and is selected fromthe group consisting of amino, halogen, hydrogen, imino, nitro, nitroso,oximido, oxo, oxy, phosphinidene, phosphino, phosphinyl,phosphinylidene, phospho, phosphono, phosphoranyl, phosphoranylidene,phosphoroso, siloxy, silyl, silylene, sulfeno, sulfinyl, sulfino, sulfo,sulfonyl, thio, and thioxo. More preferably, R^(14α) is selected fromthe group consisting of hydrogen and hydrocarbyl comprising from 1 to 6carbon atoms. Even more preferably, R^(14α) is hydrocarbyl comprisingfrom 1 to 6 carbon atoms. Most preferably, R^(14α) is ethyl.

[0100] It is especially preferred for the A ring of formula (IV) to bearomatic, as shown in formula (V):

[0101] Here, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³,X¹⁴, R^(6α), R^(6β), R^(7α), R^(7β), R^(8β), R^(9β), R^(11α), R^(11β),R^(12α), R^(12β), R^(13α, R) ^(13β), R^(14α), and R^(14β) preferably areas defined above for formula (IV).

[0102] The dashed lines are optional π bonds.

[0103] R^(6β), R^(7β), R^(8β), R^(9β), R^(11β), R^(12β), R^(13β), and/orR^(14β) are present only when X⁶, X⁷, X⁸, X⁹, X¹¹, X¹², X¹³, and/or X¹⁴,respectively, are saturated.

[0104] R^(1α) and R^(2α) preferably are independently a moiety which (a)comprises from 1 to 20 carbon atoms (more preferably from 1 to 6 carbonatoms), and is selected from the group consisting of hydrocarbyl andsubstituted hydrocarbyl; or (b) does not comprise a carbon atom and isselected from the group consisting of amino, halogen, hydrogen, nitro,nitroso, oxy, phosphino, phosphinyl, phospho, phosphono, phosphoranyl,phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and thio.More preferably, R^(1α) and R^(2α) are independently selected from thegroup consisting of hydrogen; hydrocarbyl comprising from 1 to 6 carbonatoms; and —OR¹⁰⁷, wherein R¹⁰⁷ is hydrogen or hydrocarbyl containingfrom 1 to 6 carbon atoms, and particularly wherein R¹⁰⁷ is hydrogen ormethyl. Most preferably, R^(1α) and R^(2α) are hydrogen.

[0105] R^(3α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. In a more preferred embodiment,R^(3α) is hydrogen. In another more preferred embodiment, R^(3α) isselected from the group consisting of glycosidyl, acetylated glycosidyl,and malonylated glycosidyl. In an additional more preferred embodiment,R^(3α) is —OC(O)(R¹⁰⁸), wherein R¹⁰⁸ is benzyl or —N(CH₂CH₂Cl)₂. In yetanother more preferred embodiment, R^(3α) comprises (a) no greater than20 carbon atoms (more preferably, no greater than 6 carbon atoms); and(b) a moiety selected from the group consisting of amino, imino,oximido, oxy, phosphinidene, phosphino, phosphinyl, phosphinylidene,phosphono, phosphoranyl, phosphoranylidene, siloxy, silyl, silylene,sulfeno, sulfino, sulfo, and thio. In an even more preferred embodiment,R^(3α) comprises a polarizable hydrogen atom and is, for example,—C(O)(OH), —NH₂, ═NH, ═N(OH), —OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H),—P(O)(OH)(OH), —PH₄, ═PH₃, ═SiH₂, —S(OH), —S(O)(OH), S(O)(O)(OH), or—SH. In an alternative even more preferred embodiment, R^(3α) is —OR¹⁰⁹or —OC(O)R¹¹⁰, wherein R¹⁰⁹ and R¹¹⁰ are hydrogen, halogen, orhydrocarbyl comprising from 1 to 19 carbon atoms (particularly 1 to 5carbon atoms, and more particularly 1 to 2 carbon atoms). Mostpreferably, R^(3α) is —OH.

[0106] R^(4α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 atoms), and is selected from the group consistingof hydrocarbyl and substituted hydrocarbyl; or (b) does not comprise acarbon atom and is selected from the group consisting of amino, halogen,hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl, phospho,phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino,sulfo, sulfonyl, and thio. More preferably, R^(4α) is selected from thegroup consisting of hydrogen and hydrocarbyl comprising from 1 to 6carbon atoms. Most preferably, R^(4α) is hydrogen.

[0107] In another especially preferred embodiment using the compound offormula (V) or a pharmaceutically acceptable salt thereof, no π bondsexist in the bond positions represented by both a solid line and adashed line (i.e., all the bonds in those positions are single bonds) informula (V), and the compound consequently has formula (VI) or is apharmaceutically acceptable salt thereof:

[0108] Here, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³,X¹⁴, R^(1α), R^(2α), R^(3α), R^(4α), R^(6α), R^(6β), R^(7α), R^(7β),R^(8β), R^(9β), R^(11α), R^(11β), R^(12α), R^(12β), R^(13α), R^(13β),R^(14α), and R^(14β) preferably are as defined for formula (V).

[0109] R^(6β), R^(7β), R^(11β), R^(12β), R^(13β), and R^(14β) arepresent only when X⁶, X⁷, X¹¹, X¹², X¹³, and/or X¹⁴, respectively, aresaturated.

[0110] When using the compound of formula (VI) or a pharmaceuticallyacceptable salt thereof, it is especially preferred for R^(1α), R^(2α),R^(4α), R^(6α), R^(6β), R^(7α), R^(7β), R^(8β), R^(9β), R^(11α),R^(11β), R^(12α), R^(12β), and R^(14β) to be hydrogen; R^(3α) to be —OH;R^(13α) to be —C(O)(OH)(i.e., the compound is an estrogenic carboxylicacid); R^(13β) to be methyl; and R^(14α) to be ethyl. Such a compoundmost preferably has the formula (VII) or is a pharmaceuticallyacceptable salt thereof:

[0111] In another particularly preferred embodiment, the compound offormula (V) or the pharmaceutically acceptable salt thereof has theformula (VIII) or is a pharmaceutically acceptable salt thereof:

[0112] Here, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X⁹, X¹⁰, X¹¹,X¹², X¹³, X¹⁴, R^(1α), R^(2α), R^(3α), R^(4α), R^(11α), R^(11β),R^(12α), R^(12β), R^(13α), R^(13β), R^(14α), and R^(4β) preferably areas defined for formula (VI.

[0113] R^(11β), R^(12β), R^(13β), and/or R^(14β) are present only whenX¹¹, X¹², X¹³, and/or X¹⁴, respectively, are saturated.

[0114] R^(6α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. More preferably, R^(6α) is selectedfrom the group consisting of hydrogen and hydrocarbyl comprising from 1to 6 carbon atoms. Most preferably, R^(6α) is hydrogen.

[0115] R^(7α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. More preferably, R^(7α) is selectedfrom the group consisting of hydrogen and hydrocarbyl comprising from 1to 20 carbon atoms. Most preferably, R^(7α) is hydrogen.

[0116] When the compound has formula (VIII), it is especially preferredfor R^(1α), R^(2α), R^(4α), R^(6α), R^(7α), R^(11α), R^(11β), R^(12α),R^(12β), and R^(14β) to be hydrogen; R^(3α) to be —OH; R^(13α) to be—C(O)(OH) (i.e., the compound is an estrogenic carboxylic acid); R¹³ tobe methyl; and R^(14α) to be ethyl. Such a compound, for example, mayhave the formula (IX):

[0117] This compound is sometimes described herein as“(−)-Z-bisdehydrodoisynolic acid” (or “(−)-Z-BDDA”). Its enantiomer hasthe formula (X):

[0118] This compound is sometimes described herein as“(+)-Z-bisdehydrodoisynolic acid” (or “(+)-Z-BDDA”). Depending on thetherapeutic application, either formula (IX) or formula (X) is mostpreferred.

[0119] In some instances, it is particularly preferred for R^(8α) inFormula (I) to form a carbocyclic ring with R^(7α) to form a compoundhaving the formula (XI) or a pharmaceutically acceptable salt thereof:

[0120] Here, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, R^(1α), R^(1β),R^(2α), R^(2β), R^(3α), R^(3β), R^(4α), R^(4β), R⁵, R^(6α), R^(6β),R^(7β), R^(8β), R^(9α), R^(9β), and R¹⁰ preferably are as defined abovefor formula (I).

[0121] R^(1β), R^(2β), R^(3β), R^(4β), R⁵, R^(6β), R^(7β), R^(8β),R^(9β), R¹⁰, R^(14β), R^(20β), R^(21β), and/or R^(22β), respectively,are saturated.

[0122] R^(21α) and R^(22α) preferably are independently a moiety which(a) comprises from 1 to 20 carbon atoms (more preferably, from 1 to 6carbon atoms), and is selected from the group consisting of hydrocarbyland substituted hydrocarbyl; or (b) does not comprise a carbon atom andis selected from the group consisting of amino, halogen, hydrogen,imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More preferably,R^(21α) and R^(22α) are independently selected from the group consistingof hydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms. Mostpreferably, R^(21α) and R^(22α) are hydrogen.

[0123] R^(21β), R^(22β), and R^(14β) preferably are independently amoiety which (a) comprises from 1 to 20 carbon atoms (more preferably,from 1 to 6 carbon atoms), and is selected from the group consisting ofhydrocarbyl and substituted hydrocarbyl; or (b) does not comprise acarbon atom and is selected from the group consisting of amino, halogen,hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl, phospho,phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino,sulfo, sulfonyl, and thio. More preferably, R^(21β), R^(22β), andR^(14β) are independently selected from the group consisting of hydrogenand hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,R^(21β), R^(22β), and R^(14β) are hydrogen.

[0124] R^(20α) preferably comprises (a) no greater than 20 carbon atoms(more preferably no greater than 6 carbon atoms); and (b) a moietyselected from the group consisting of amino, imino, oximido, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phosphono,phosphoranyl, phosphoranylidene, siloxy, silyl, silylene, sulfeno,sulfino, sulfo, and thio. More preferably, R^(20α) comprises apolarizable hydrogen atom and is, for example, —C(O)(OH), —NH₂, ═NH,═N(OH), —OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H), —P(O)(OH)(OH), —PH₄,═PH₃, ═SiH₂, —S(OH), —S(O)(OH), S(O)(O)(OH), or —SH. In an alternativemore preferred embodiment, R^(20α) is —C(O)(OR¹¹¹), wherein R¹¹¹ ishydrogen, halogen, or hydrocarbyl comprising from 1 to 5 carbon atoms.Most preferably, R^(20α) is —C(O)(OH) (i.e., the compound is anestrogenic carboxylic acid).

[0125] R^(20β) preferably (a) comprises from 1 to 20 carbon atoms and isselected from the group consisting of hydrocarbyl and substitutedhydrocarbyl; or (b) does not comprise a carbon atom and is selected fromthe group consisting of amino, halogen, hydrogen, nitro, nitroso, oxy,phosphino, phosphinyl, phospho, phosphono, phosphoranyl, phosphoroso,siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and thio. Morepreferably, R^(20β) is selected from the group consisting of hydrogenand hydrocarbyl comprising from 1 to 6 carbon atoms. Even morepreferably, R^(20β) is hydrocarbyl comprising from 1 to 6 carbon atoms.Most preferably, R^(20β) is methyl.

[0126] R^(14α) preferably (a) comprises from 1 to 20 carbon atoms and isselected from the group consisting of hydrocarbyl and substitutedhydrocarbyl; or (b) does not comprise a carbon atom and is selected fromthe group consisting of amino, halogen, hydrogen, imino, nitro, nitroso,oximido, oxo, oxy, phosphinidene, phosphino, phosphinyl,phosphinylidene, phospho, phosphono, phosphoranyl, phosphoranylidene,phosphoroso, siloxy, silyl, silylene, sulfeno, sulfinyl, sulfino, sulfo,sulfonyl, thio, and thioxo. More preferably, R^(14α) is selected fromthe group consisting of hydrogen and hydrocarbyl comprising from 1 to 6carbon atoms. Even more preferably, R^(14α) is hydrocarbyl comprisingfrom 1 to 6 carbon atoms. Most preferably, R^(14α) is ethyl.

[0127] When the compound has formula (XI), it is particularly preferredfor the A and B rings to be aromatic; R^(1α), R^(2α), R^(4α), R^(6α),R^(9α), R^(14β), R^(21α), R^(21β), R^(22α), and R^(2β) to be hydrogen;R^(3α) to be —OH; R^(14α), to be ethyl; R^(20α) to be —C(O)(OH) (i.e.,the compound is an estrogenic carboxylic acid); R^(20β) to be methyl.Most preferably, such a compound is formula (XII) or a pharmaceuticallyacceptable salt thereof:

[0128] This compound is sometimes referred to herein as1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2-carboxylicacid.

[0129] In another of the more preferred embodiments of this invention,the compound has the structure of formula (XIII) or is apharmaceutically acceptable salt thereof:

[0130] Here, X¹, X², X³, X⁴, X⁵, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, and X¹⁴preferably are carbon atoms.

[0131] The dashed lines are optional π bonds.

[0132] R^(1β), R^(2β), R^(3β), R^(4β), R^(5β), R^(8β), R⁹, R¹⁰, R^(11β),R^(12β), R^(13β), and/or R^(14β) are present only when X¹, X², X³, X⁴,X⁵, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, and/or X¹⁴, respectively, are saturated.

[0133] R^(1α) and R^(2α) preferably are independently a moiety which (a)comprises from 1 to 20 carbon atoms (more preferably from 1 to 6 carbonatoms), and is selected from the group consisting of hydrocarbyl andsubstituted hydrocarbyl; or (b) does not comprise a 10 carbon atom andis selected from the group consisting of amino, halogen, hydrogen,imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More preferably,R^(1α) and R^(2α) are independently selected from the group consistingof hydrogen; hydrocarbyl 15 comprising from 1 to 6 carbon atoms; and—OR¹¹², wherein R¹¹² is hydrogen or hydrocarbyl containing from 1 to 6carbon atoms, and particularly wherein R¹¹² is hydrogen or methyl. Mostpreferably, R^(1α) and R^(2α) are hydrogen.

[0134] R^(1β), R^(2β), R^(3β), R^(4β), R^(5β), R^(8β), R⁹, R¹⁰, R^(11β),and R^(13β) preferably are independently a moiety which (a) comprisesfrom 1 to 20 carbon atoms (more preferably from 1 to 6 carbon atoms),and is selected from the group consisting of hydrocarbyl and substitutedhydrocarbyl; or (b) does not comprise a carbon atom and is selected fromthe group consisting of amino, halogen, hydrogen, nitro, nitroso, oxy,phosphino, phosphinyl, phospho, phosphono, phosphoranyl, phosphoroso,siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and thio. Morepreferably, R^(1β), R^(2β), R^(3β), R^(4β), R^(5β), R^(8β), R⁹, R¹⁰,R^(11β), and R^(13β) are independently selected from the groupconsisting of hydrogen and hydrocarbyl comprising from 1 to 6 carbonatoms. Most preferably, R^(1β), R^(2β), R^(3β), R^(4β), R^(5β), R^(8β),R⁹, R¹⁰, R^(11β), and R^(13β) are hydrogen.

[0135] R^(3α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, imino, nitro, nitroso, oximido, oxo, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl,silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo.In a more preferred embodiment, R^(3α) is hydrogen. In another morepreferred embodiment, R^(3α) is selected from the group consisting ofglycosidyl, acetylated glycosidyl, and malonylated glycosidyl. In anadditional more preferred embodiment, R^(3α) is —OC(O)(R¹¹⁴), whereinR¹¹⁴ is benzyl or —N(CH₂CH₂Cl)₂. In yet another more preferredembodiment, R^(3α) comprises (a) no greater than 20 carbon atoms (morepreferably, no greater than 6 carbon atoms); and (b) a moiety selectedfrom the group consisting of amino, halogen, hydrogen, imino, oximido,oxo, oxy, phosphinidene, phosphino, phosphinyl, phosphinylidene,phosphono, phosphoranyl, phosphoranylidene, siloxy, silyl, silylene,sulfeno, sulfino, sulfo, and thio. In an even more preferred embodiment,R^(3α) comprises (a) no greater than 20 carbon atoms (more preferably,no greater than 6 carbon atoms); and (b) a moiety selected from thegroup consisting of amino, imino, oximido, oxy, phosphinidene,phosphino, phosphinyl, phosphinylidene, phosphono, phosphoranyl,phosphoranylidene, siloxy, silyl, silylene, sulfeno, sulfino, sulfo, andthio. In a still even more preferred embodiment, R^(3α) comprises apolarizable hydrogen atom, and is, for example, —C(O)(OH), —NH₂, ═NH,═N(OH), —OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H), —P(O)(OH)(OH), —PH₄,═PH₃, ═SiH₂, —S(OH), —S(O)(OH), S(O)(O)(OH), or —SH. In an alternativeeven more preferred embodiment, R^(3α) is —OR¹¹⁵ or —OC(O)R¹¹⁶, whereinR¹¹⁵ and R¹¹⁶ are hydrogen, halogen, or hydrocarbyl comprising from 1 to19 carbon atoms (particularly 1 to 5 carbon atoms, and more particularlyfrom 1 to 2 carbon atoms). Most preferably, R^(3α) is —OH.

[0136] R^(4α), R^(5α), and R^(11α) preferably are independently a moietywhich (a) comprises from 1 to 20 carbon atoms (more preferably from 1 to6 carbon atoms), and is selected from the group consisting ofhydrocarbyl and substituted hydrocarbyl; or (b) does not comprise acarbon atom and is selected from the group consisting of amino, halogen,hydrogen, imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene,phosphino, phosphinyl, phosphinylidene, phospho, phosphono,phosphoranyl, phosphoranylidene, phosphoroso, siloxy, silyl, silylene,sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. Morepreferably, R^(4α), R^(5α), and R^(11α) are independently selected fromthe group consisting of hydrogen and hydrocarbyl comprising from 1 to 6carbon atoms. Most preferably, R^(4α), R^(5α), and R^(11α) are hydrogen.

[0137] R^(8α), R^(12α), and R^(14α) preferably are independently amoiety which (a) comprises from 1 to 20 carbon atoms and is selectedfrom the group consisting of hydrocarbyl and substituted hydrocarbyl; or(b) does not comprise a carbon atom and is selected from the groupconsisting of amino, halogen, hydrogen, imino, nitro, nitroso, oximido,oxo, oxy, phosphinidene, phosphino, phosphinyl, phosphinylidene,phospho, phosphono, phosphoranyl, phosphoranylidene, phosphoroso,siloxy, silyl, silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl,thio, and thioxo. More preferably, R^(8α), R^(12α), and R^(14α) areindependently selected from the group consisting of hydrogen andhydrocarbyl comprising from 1 to 6 carbon atoms (particularly from 1 to2 carbon atoms).

[0138] R^(13α) preferably comprises (a) no greater than 20 carbon atoms(more preferably no greater than 6 carbon atoms); and (b) a moietyselected from the group consisting of amino, imino, oximido, oxy,phosphinidene, phosphino, phosphinyl, phosphinylidene, phosphono,phosphoranyl, phosphoranylidene, siloxy, silyl, silylene, sulfeno,sulfino, sulfo, and thio. More preferably, R^(13α) comprises apolarizable hydrogen atom and is, for example, —C(O)(OH), —NH₂, ═NH,═N(OH), —OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H), —P(O)(OH)(OH), —PH₄,═PH₃, ═SiH₂, —S(OH), —S(O)(OH), S(O)(O)(OH), or —SH. In an alternativemore preferred embodiment, R^(13α) is —C(O)(OR¹¹⁷), wherein R¹¹⁷ ishydrogen, halogen, or hydrocarbyl comprising from 1 to 5 carbon atoms.Most preferably, R^(13α) is —C(O)(OH) (i.e., the compound is anestrogenic carboxylic acid).

[0139] R^(12β) and R^(14β) preferably are independently a moiety which(a) comprises from 1 to 20 carbon atoms and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. More preferably, R^(12β) and R^(14β)are independently selected from the group consisting of hydrogen andhydrocarbyl comprising from 1 to 6 carbon atoms (particularly from 1 to2 carbon atoms).

[0140] When the compound has the structure of formula (XIII) or is apharmaceutically acceptable salt thereof, it is particularly preferredfor the compound to have formula (XIV) or be a pharmaceuticallyacceptable thereof:

[0141] Here, X¹, X², X³, X⁴, X⁵, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴,R^(8α), R^(8β), R⁹, R^(11α), R^(11β), R^(12α), R^(12β), R^(13α),R^(13β), R^(14α), and R^(14Γ) preferably are as defined as in formula(XIII).

[0142] The dashed lines are optional π bonds.

[0143] R^(8β), R⁹, R^(12β), R^(13β), and/or R^(14β) are present onlywhen X⁸, X⁹, X¹¹, X¹², X¹³, and/or X¹⁴, respectively, are saturated.

[0144] R^(1α) and R^(2α) preferably are independently a moiety which (a)comprises from 1 to 20 carbon atoms (more preferably from 1 to 6 carbonatoms), and is selected from the group consisting of hydrocarbyl andsubstituted hydrocarbyl; or (b) does not comprise a carbon atom and isselected from the group consisting of amino, halogen, hydrogen, nitro,nitroso, oxy, phosphino, phosphinyl, phospho, phosphono, phosphoranyl,phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and thio.More preferably, R^(1α) and R^(2α) are independently selected from thegroup consisting of hydrogen; hydrocarbyl comprising from 1 to 6 carbonatoms; and —OR¹¹⁸, wherein R¹¹⁸ is hydrogen or hydrocarbyl containingfrom 1 to 6 carbon atoms, and particularly wherein R¹¹⁸ is hydrogen ormethyl. Most preferably, R^(1α) and R^(2α) are hydrogen.

[0145] R^(3α) preferably (a) comprises from 1 to 20 carbon atoms (morepreferably from 1 to 6 carbon atoms), and is selected from the groupconsisting of hydrocarbyl and substituted hydrocarbyl; or (b) does notcomprise a carbon atom and is selected from the group consisting ofamino, halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl, sulfeno,sulfino, sulfo, sulfonyl, and thio. In a more preferred embodiment,R^(3α) is hydrogen. In another more preferred embodiment, R^(3α) isselected from the group consisting of glycosidyl, acetylated glycosidyl,and malonylated glycosidyl. In an additional more preferred embodiment,R^(3α) is —OC(O)(R¹¹⁹), wherein R¹¹⁹ is benzyl or —N(CH₂CH₂Cl)₂. In yetanother more preferred embodiment, R^(3α) comprises (a) no greater than20 carbon atoms (more preferably, no greater than 6 carbon atoms); and(b) a moiety selected from the group consisting of amino, imino,oximido, oxy, phosphinidene, phosphino, phosphinyl, phosphinylidene,phosphono, phosphoranyl, phosphoranylidene, siloxy, silyl, silylene,sulfeno, sulfino, sulfo, and thio. In an even more preferred embodiment,R^(3α) comprises a polarizable hydrogen atom and is, for example,—C(O)(OH), —NH₂, ═NH, ═N(OH), —OH, ═PH, —PH₂, —P(O)(H)(H), ═P(O)(H),—P(O)(OH)(OH), —PH₄, ═PH₃, ═SiH₂, —S(OH), —S(O)(OH), S(O)(O)(OH), or—SH. In an alternative even more preferred embodiment, R^(3α) is —OR¹²⁰or —OC(O)R¹²¹, wherein R¹²⁰ and R¹²¹ are hydrogen, halogen, orhydrocarbyl comprising from 1 to 19 carbon atoms (particularly 1 to 5carbon atoms, and more particularly 1 to 2 carbon atoms). Mostpreferably, R^(3α) is —OH.

[0146] R^(4α) and R^(5α) preferably are independently a moiety which (a)comprises from 1 to 20 carbon atoms (more preferably from 1 to 6 atoms),and is selected from the group consisting of hydrocarbyl and substitutedhydrocarbyl; or (b) does not comprise a carbon atom and is selected fromthe group consisting of amino, halogen, hydrogen, nitro, nitroso, oxy,phosphino, phosphinyl, phospho, phosphono, phosphoranyl, phosphoroso,siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and thio. Morepreferably, R^(4α) and R^(5α) are independently selected from the groupconsisting of hydrogen and hydrocarbyl comprising from 1 to 6 carbonatoms. Most preferably, R^(4α) and R^(5α) are hydrogen.

[0147] Examples of preferred compounds having the structure of formula(XIII) include the following estrogenic carboxylic acids (andpharmacuetically acceptable salts thereof):

[0148] 1. 1-(p-hydroxyphenyl)-6-ethyl-5-methylcyclohexene-4-carboxylicacid:

[0149] 2. 1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohexene-4-carboxylicacid:

[0150] 3.1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohexene-4-carboxylicacid:

[0151] 4. 4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexane carboxylicacid:

[0152] 5. 1-phenyl-2-ethyl-3-methylcyclohexene-4-carboxylic acid:

[0153] 6. 1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid:

[0154] For a review of the structures of estrogenic doisynolic-typeacids, and methods for preparing these compounds, the reader is referredto the review of Meyers et al., “Doisynolic-Type Acids-UterotropicallyPotent Estrogens Which Compete Poorly With Estradiol for CytosolicEstradiol Receptors, J. Steroid Biochem. 31(4A):393-404 (1988), and thereferences cited therein.

[0155] B. Pharmaceutical Compositions

[0156] The compounds of the present invention can be formulated aspharmaceutical compositions. Such compositions can be administeredorally, parenterally, by inhalation spray, rectally, intradermally,transdermally, or topically in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired. Topical administration may also involve the useof transdermal administration such as transdermal patches oriontophoresis devices. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, or intrastemal injection, orinfusion techniques. Formulation of drugs is discussed in, for example,Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pennsylvania (1975), and Liberman, H. A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).

[0157] Injectable preparations, for example, sterile injectable aqueousor oleaginous suspensions, can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid are usefulin the preparation of injectables. Dimethyl acetamide, surfactantsincluding ionic and non-ionic detergents, and polyethylene glycols canbe used. Mixtures of solvents and wetting agents such as those discussedabove are also useful.

[0158] Suppositories for rectal administration of the compoundsdiscussed herein can be prepared by mixing the active agent with asuitable non-irritating excipient such as cocoa butter, synthetic mono-,di-, or triglycerides, fatty acids, or polyethylene glycols which aresolid at ordinary temperatures but liquid at the rectal temperature, andwhich will therefore melt in the rectum and release the drug.

[0159] Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, thecompounds of this invention are ordinarily combined with one or moreadjuvants appropriate to the indicated route of administration. Ifadministered per os, the compounds can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents such as sodium citrate, or magnesiumor calcium carbonate or bicarbonate. Tablets and pills can additionallybe prepared with enteric coatings.

[0160] For therapeutic purposes, formulations for parenteraladministration can be in the form of aqueous or non-aqueous isotonicsterile injection solutions or suspensions. These solutions andsuspensions can be prepared from sterile powders or granules having oneor more of the carriers or diluents mentioned for use in theformulations for oral administration. The compounds can be dissolved inwater, polyethylene glycol, propylene glycol, ethanol, corn oil,cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride,and/or various buffers. Other adjuvants and modes of administration arewell and widely known in the pharmaceutical art.

[0161] Liquid dosage forms for oral administration can includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs containing inert diluents commonly used in the art, such aswater. Such compositions can also comprise adjuvants, such as wettingagents, emulsifying and suspending agents, and sweetening, flavoring,and perfuming agents.

[0162] The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the patient and the particular mode of administration.

[0163] The mode of administration is partially dependent upon thechemical form of the estrogenic carboxylic acids. The phenolic andcarboxylic salts (e.g., sodium, potassium, calcium, etc.) are more watersoluble than the parent phenolic carboxylic acids, and can beadministered orally or in aqueous solution. The estrogenic carboxylicacids themselves and their esters and related derivatives have lowerwater solubility and are probably best administered subcutaneously ortransdermally in an oily or penetrating vehicle.

[0164] In the form of their 3-methyl ethers, (±)-Z-BDDA (“Fenocylin”,Ciba-Geigy) and allenolic acid (“Vallestril”, G. D. Searle and Co.) havebeen cleared for clinical use. However, Segaloff ((1949) in RecentProgress in Hormone Research, Vol. IV, G. Pincus, Ed., Academic Press,New York, pp. 85-111) discounted the clinical activity of Fenocylin inwomen. See Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos C F, DandlikerW B: “Doisynolic-Type Acids—Uterotropically Potent Estrogens whichCompete Poorly with Estradiol for Cytosolic Estradiol Receptors. JSteroid Biochem 31:393-404 (1988); and Soto A M, Meyers C Y,Sonnenschein C: “How Many Rings Can be Cleaved from a Steroidal EstrogenWhile Preserving its Estrogenic Activity?“The Endocrine Society, 70thAnnual Meeting, Abstract (1988); and the foregoing discussion.

[0165] Certain of the pharmaceutical compounds of this invention whichare administered in accordance with the methods of the invention canserve as prodrugs to other compounds of this invention. Prodrugs aredrugs that can be chemically converted in vivo or in vitro by biologicalsystems into an active derivative or derivatives. Prodrugs areadministered in essentially the same fashion as the other pharmaceuticalcompounds of the invention. Non-limiting examples includenon-hydroxylated phenylcyclohexenecarboxylic acids of this inventionthat are hydroxylated in vivo.

[0166] It should be noted that the present invention encompasses the useof estrogenic carboxylic acids as disclosed herein formulated alone, andin various combinations with one another. Single estrogenic carboxylicacids, or combinations of estrogenic carboxylic acids, can also beformulated in combination with other estrogens coventionally used in theart.

[0167] C. Dosages

[0168] Depending upon the particular pharmaceutical application, theestrogenically active compounds of the present invention can beadministered daily to humans or animals in a number of differentdosages. For example, as suggested by the results disclosed in Example2, below, the dosage can be an amount in the range of from about 0.1μg/kg/day to about 100 mg/kg/day, preferably from about 0.5 μg/kg/day toabout 75 mg/kg/day, more preferably from about 1 μg/kg/day to about 50mg/kg/day, even more preferably from about 1 μg/kg/day to about 25mg/kg/day, and still more preferably from about 1 μg/kg/day to about 20mg/kg/day. As suggested by the results disclosed in Example 4, below,dosages for use in treating prostatic (and other) disorders can be anamount in the range of from about 10 μg/kg/day to about 10 mg/kg/day,preferably from about 10 μg/kg/day to about 5 mg/kg/day, more preferablyfrom about 10 μg/kg/day to about 2.5 mg/kg/day, and even more preferablyfrom about 10 μg/kg/day to about 1 mg/kg/day. In further embodiments,the lower value of these dosage ranges can be as low as about 1μg/kg/day.

[0169] The doses described above can be administered to a patient in asingle dose or in proportionate multiple subdoses, for example twosubdoses daily. In the case of proportionate multiple subdoses, dosageunit compositions can contain such amounts of submultiples thereof tomake up the daily dose. Multiple doses per day can also increase thetotal daily dose should this be desired by the person prescribing thedrug.

[0170] D. Treatment Regimen

[0171] The present invention provides methods for treating or preventinga variety of symptoms, conditions, and diseases that would benefit fromestrogen therapy using the compounds disclosed herein. In this context,“treating” refers to ameliorating, suppressing, or eradicating thesesymptoms, conditions, and diseases. The regimen for treating a patientsuffering from a symptom, condition, or disease that would benefit fromestrogen therapy, or preventing the same, with the compounds and/orcompositions of the present invention is selected in accordance with avariety of factors, including the age, weight, sex, diet, and medicalcondition of the patient, the severity of the infection, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetic, and toxicology profiles of the particularcompounds employed, and whether a drug delivery system is utilized. Itshould be noted that the methods disclosed herein are applicable in bothhuman and veterinary medicine. Treatment of domestic pets, such as catsand dogs, is contemplated in the present invention.

[0172] Administration of individual estrogenic carboxylic acids,combinations thereof, or such individual estrogenic carboxylic acids orcombinations thereof in further combination with estrogensconventionally used in the art, should generally be continued over aperiod of several weeks to several months or years until symptoms reachacceptable levels, or have been eliminated entirely, indicating that thecondition has been controlled or eradicated. As noted above, patientsundergoing treatment with the drugs disclosed herein can be routinelymonitored by measuring appropriate physical and physiological parametersto determine the effectiveness of therapy.

[0173] Continuous analysis of the data obtained by these methods permitsmodification of the treatment regimen during therapy so that optimalamounts of each compound are administered, and so that the duration oftreatment can be determined as well. Thus, the treatment regimen/dosingschedule can be rationally modified over the course of therapy so thatthe lowest amount of each estrogenic carboxylic acid used alone or incombination which together exhibit satisfactory therapeuticeffectiveness are administered, and so that administration of suchcompounds is continued only so long as is necessary to successfullytreat the indicated condition.

[0174] In order to monitor the effect and progress of treatment,conventional assays can be used wherever appropriate. For example, thestandard immunoassays for testosterone and prostate specific antigen(PSA) can be used in the case of prostate cancer. Significant decreasesin either testosterone or PSA indicate the utility of the presentcompounds as therapeutic agents. When such assays are lacking or whereeffects are expected to be very slow, more subjective parameters can beemployed. These are considered individually in the following examplesdiscussing each disease.

[0175] The chronic effects of the Z-BDDA compounds as compared to thoseof E2 were studied in rats as a model mammalian system. In addition toanlysis of changes in body weight, additional metabolic and endocrinestudies were performed, including monitoring food intake and metabolicand reproductive parameters in male and female rats. Because so littlehas been reported on the comparative effects of the Z-BDDA enantiomers,the (+)-, (−)-, and (±)-forms were prepared and investigated. Anner G,Miescher K: Hydrierungs-Und Umlagerungs-Reaktion in derDoisynolsäure-Reihe. Oestrogene Carbonsäuren XII. Helv. Chim. Acta 29(1946) 1889-1895; Die totalsyntheses von racemischen doisynolsäuren XXI.Über oestrogene carbonsäueren. ibid 30:1422-1432 (1947); Tschopp E:“Wirksamkeit, organconzentration und ausscheidung der7-methyl-bisdehydro-doisynolsäure.” Helv Physiol Pharmacol Acta4:401-410 (1946); Tschopp E: “Die oestrogene wirkung derbisdehydrodoisynolsäure und ihre derivate.” Helv Physiol Pharmacol Acta4:271-284 (1946); Rometsch R, Miescher K: “Die spaltung des racematesder n-bisdehydro-doisynolsäure. Über östrogene carbonsäuren X.” HelvChim Acta 29:1231-1235 (1946); and Terenius L: “Differential InhibitionIn Vitro of 17β-Estradiol Binding in the Mouse Uterus and Vagina byOptical Antipodes of Estrogen.” Molec Pharmac 4:301-310 (1968).

[0176] Definitions

[0177] The term “acyl” means the group having the formula —C(O)(R),wherein R is hydrocarbyl. The term “substituted acyl” means the grouphaving the formula —C(O)(R), wherein R is, for example, substitutedhydrocarbyl.

[0178] The term “alkanoyl halide” means the group having the formula—C(O)(R), wherein R is halogen.

[0179] The term “alkenyl” means a straight or branched hydrocarbylcomprising at least one carbon-carbon double bond, and includes, forexample, ethenyl, propenyl, iso-propenyl, butenyl, isobutenyl, hexenyl,and the like.

[0180] The term “alkyl” means a saturated straight or branched chainhydrocarbyl (i.e., no double or triple carbon-carbon bonds), andincludes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.

[0181] The term “alkynyl” means a straight or branched hydrocarbylcomprising at least one triple carbon-carbon bond, and includes, forexample, ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.

[0182] The term “amide” means the group having the formula—C(O)(N(R^(a))(R^(b))), wherein R^(a) and R^(b) are independently, forexample, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0183] The term “amino” means the group having the formula—N(R^(a))(R^(b)), wherein R^(a) and R^(b) are independently, forexample, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0184] The term “carboxyl” means the group having the formula —C(O)(OR),wherein R is, for example, hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

[0185] The term “formyl” means the group having the formula —C(H)(O).

[0186] The term “halogen” includes F, Cl, Br, and I.

[0187] The term “heterocyclyl” means a chain of 3 or more atoms(typically 5 or 6 atoms) forming a ring (or multiple rings), wherein atleast one of the atoms forming the ring is an atom which is not a carbonatom or hydrogen atom (e.g., sulfur, nitrogen, or oxygen). Theheterocyclyl may comprise all single bonds between the atoms forming thering, or, alternatively, may comprise one or more double bonds betweensuch atoms. Heterocyclyls include, for example, furyl, thienyl,pyridinyl, morpholinyl, and the like. In addition to being bound to theother atoms forming the ring, the atoms forming the ring of theheterocyclyl may also be bound to hydrogen or to another group, such as,for example: (a) a group which is selected from the group consisting ofhydrocarbyl or a substituted hydrocarbyl (e.g., another heterocyclyl);or (b) a group which does not comprise a carbon atom and is selectedfrom the group consisting of an amino, halogen, hydrogen, imino, nitro,nitroso, oximido, oxo, oxy, phosphinidene, phosphino, phosphinyl,phosphinylidene, phospho, phosphono, phosphoranyl, phosphoranylidene,phosphoroso, siloxy, silyl, silylene, sulfeno, sulfino, sulfinyl, sulfo,sulfonyl, thio, and thioxo.

[0188] The term “hydrocarbyl” means a group consisting exclusively ofcarbon and hydrogen atoms. Such a group may be straight, branched,cyclic (or multi-cyclic), or a combination thereof. It may also besaturated (i.e., comprise no carbon-carbon double or triple bonds) orunsaturated (i.e., comprise at least one carbon-carbon double or triplebond). Hydrocarbyls include, for example, alkyl, alkenyl, alkynyl, aryl,alkaryl, alkenaryl, and alkynaryl. The term “substituted hydrocarbyl”means a hydrocarbyl, wherein at least one hydrogen atom has beensubstituted with (a) an atom which is not a hydrogen or carbon atom(i.e., a heteroatom), or (b) a group of atoms comprising at least oneheteroatom. A “heteroatom” may be, for example, a boron, halogen,nitrogen, oxygen, phosphorous, silicon, or sulfur atom. Substitutedhydrocarbyls include hydrocarbyls wherein one or more hydrogen atomshave been substituted with, for example, amino, halogen, heterocyclyl,imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,sulfinyl, sulfino, sulfo, sulfonyl, thio, or thioxo. Examples ofsubstituted hydrocarbyls include acyl (e.g., acetyl and benzoyl) andsubstituted acyl, alkanoyl halide, amide, formyl, nitrile, carboxyl,oxycarbonyl, alkoxy, amino substituted with hydrocarbyl (i.e.,N(R^(a))(R^(b)), wherein R^(a) and R^(b) are hydrocarbyl), phosphonosubstituted with hydrocarbyl (i.e., a phosphono ester,—P(O(OR^(a))(OR^(b)), wherein R^(a) and R^(b) are hydrocarbyl), andsulfo substituted with hydrocarbyl (i.e., a sulfo ester, —S(O)(O)(OR),wherein R is hydrocarbyl).

[0189] The term “imino” means the group having the formula═NR, wherein Ris, for example, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0190] The term “nitrile” means the group having the formula —CN.

[0191] The term “nitro” means the group having the formula —NO₂.

[0192] The term “nitroso” means the group having the formula —NO.

[0193] The term “non-hydrocarbyl group” means a group that comprises nocarbon atoms.

[0194] The term “oximido” means the group having the formula ═N(OR),wherein R is, for example, hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

[0195] The term “oxo” means the oxygen group (i.e., ═O) of a carbonylgroup.

[0196] The term “oxy” means the group having the formula —OR, wherein Ris, for example, hydrogen (i.e., —OR is hydroxy), hydrocarbyl, orsubstituted hydrocarbyl.

[0197] The term “oxycarbonyl” means the group having the formula—OC(O)(R), wherein R is, for example, hydrocarbyl or substitutedhydrocarbyl.

[0198] The term “pharmaceutically acceptable salt” embraces saltscommonly used to form alkali metal salts and to form addition salts offree acids or free bases. The nature of the salt is not critical,provided that it is pharmaceutically-acceptable. Suitablepharmaceutically-acceptable acid addition salts of the therapeuticcompounds discussed herein may be prepared from an inorganic acid orfrom an organic acid. Examples of such inorganic acids are hydrochloric,hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclyl, carboxylic andsulfonic classes of organic acids, example of which are formic, acetic,propionic, succinic, glycolic, gluconic, lactic, malic, tartaric,citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic,glutamic, benzoic, anthranilic, mesylic, stearic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic,cyclohexylaminosulfonic, algenic, b-hydroxybutyric, galactaric andgalacturonic acid. Suitable pharmaceutically-acceptable base additionsalts of the therapeutic compounds discussed herein include metallicsalts and organic salts. More preferred metallic salts include, but arenot limited to, appropriate alkali metal (group Ia) salts, alkalineearth metal (group IIa) salts, and other physiological acceptablemetals. Such salts can be made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc. Preferred organic salts can bemade from tertiary amines and quaternary ammonium salts, including inpart, tromethamine, diethylamine, N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine) and procaine. All of these salts may be prepared byconventional means from the corresponding therapeutic compoundsdiscussed herein by reacting, for example, the appropriate acid or basewith the compounds.

[0199] The term “phosphinidene” means the group having the formula ═PR,wherein R is, for example, hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

[0200] The term “phosphino” means the group having the formula—P(R^(a))(R^(b)), wherein R^(a) and R^(b) are independently, forexample, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0201] The term “phosphinyl” means the group having the formula—P(O)(R^(a))(R^(b)), wherein R^(a) and R^(b) are independently, forexample, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0202] The term “phosphinylidene” means the group having the formula═P(O)(R), wherein R is, for example, hydrogen, hydrocarbyl, orsubstituted hydrocarbyl.

[0203] The term “phospho” means the group having the formula —PO₂.

[0204] The term “phosphono” means the group having the formula—P(O)(OR^(a))(OR^(b)), wherein R^(a) and R^(b) are independently, forexample, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0205] The term “phosphoranyl” means the group having the formula—P(R^(a))(R^(b))(RC)(Rd), wherein R^(a), R^(b), R^(c), and R^(d) areindependently, for example, hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

[0206] The term “phosphoranylidene” means the group having the formula═P(R^(a))(R^(b))(R^(c)), wherein R^(a), R^(b), and R^(c) areindependently, for example, hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

[0207] The term “phosphoroso” means the group having the formula —PO.

[0208] The term “siloxy” means the group having the formula—OSi(R^(a))(R^(b))(R^(c)), wherein R^(a), R^(b), and R^(c) areindependently, for example, hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

[0209] The term “silyl” means the group having the formula—Si(R^(a))(R^(b))(c), wherein R^(a), R^(b), and R^(c) are independently,for example, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0210] The term “silylene” means the group having the formula═Si(R^(a))(R^(b)), wherein R^(a) and R^(b) are independently, forexample, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0211] The term “unsubstituted silylene” means the group having theformula ═SiH₂.

[0212] The term “sulfeno” means the group having the formula —S(OR),wherein R is, for example, hydrocarbyl or substituted hydrocarbyl.

[0213] The term “sulfino” means the group having the formula —S(O)(OH).The term “substituted sulfino” means the group having the formula—S(O)(OR), wherein R is, for example, hydrocarbyl or substitutedhydrocarbyl.

[0214] The term “sulfinyl” means the group having the formula ═SO.

[0215] The term “sulfo” means the group having the formula —S(O)(O)(OR),wherein R is, for example, hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

[0216] The term “sulfonyl” means the group having the formula—S(O)(O)(R), wherein R is, for example, halogen, hydrocarbyl,substituted hydrocarbyl, or amine.

[0217] The term “thio” means the group having the formula —SR, wherein Ris, for example, hydrogen, hydrocarbyl, or substituted hydrocarbyl.

[0218] The term “thioxo” means the group having the formula ═S.

EXAMPLES

[0219] The following non-limiting examples illustrate various aspects ofthe present invention.

Example 1 Preparation of (±-Z-bis dehydrodoisynolic acid ((±)-Z-BDDA))

[0220] (±)-Z-bisdehydrodoisynolic acid ((±)-Z-BDDA)) is prepared byrefluxing a solution of Fenocylin in concentrated HBr-HOAc for 2.5 hr.After recrystallization, the melting point is 204-205.5° C. Elementalanalysis, acid-base titration, and NMR can be used to confirm theidentify of the product.

[0221] Alternatively, potassium hydroxide fusion of equilenin yields amixture of acids from which the (−)-Z-bis-dehydrodoisynolic acid can beisolated (K. Miescher, Chem. Rev. 43:367-384 (1948)). One or more of theallenolic acids can be prepared by literature methods cited in Miescher.The other non-steroidal estrogenic compounds of the present invention,i.e., the hydroxyphenylcyclohexane-and -cyclo-hexene-, andhydroxytetrahydroanthracenecarboxylic acids disclosed herein, can besynthesized by methods disclosed in references discussed in Meyers etal., J. Steroid Biochem. 31(4A):393-404 (1988).

Example 2 Comparative Effects of (−)-, (+)-, and(±)-Z-bisdehydrodoisynolic Acids and Estradiol on Body Weight, FoodIntake, Metabolic, and Reproductive Parameters in Male and Female Rats

[0222] A study was designed to investigate the chronic effects of theZ-BDDA compounds vs. E2 in rats. In addition to analysis of changes inbody weight, additional metabolic and endocrine studies were performed,including monitoring food intake and metabolic and reproductiveparameters, in male and female rats. Moreover, because so little hasbeen reported on the comparative effects of the Z-BDDA enantiomers, the(+), (−) and (±) forms were prepared and investigated.

[0223] The compounds used in these studies were (+)-17β-estradiol (E2),(−)-Z-bisdehydrodoisynolic acid [(−)-Z-BDDA], and(+)-Z-bisdehydrodoisynolic acid [(±)-Z-BDDA]. Their structures are shownbelow in a, b, and c, respectively. Racemic (±)-Z-bisdehydrodoisynolicacid [(±)-Z-BDDA] is a 1:1 mixture of the (+) and the (−) enantiomers.

[0224] (+)-17β-Estradiol (E2) was purchased from Sigma Chemical Company(St. Louis, Mo.). (±)-Z-Bisdehydrodoisynolic acid [((±)-Z-BDDA] wasprepared from (±)-Z-BDDA-3-OMe (“Fenocylin,” from Ciba-Geigy, Inc.; m.p.228-230° C) as described by Meyers et al. (Meyers C Y, Kolb V M, Gass GH, Rao B R, Roos C F, Dandliker W B: “Doisynolic-TypeAcids-Uterotropically Potent Estrogens which Compete Poorly withEstradiol for Cytosolic Estradiol Receptors. J Steroid Biochem31:393-404 (1988); and Banz J, Winters T A, Hou Y, Adler S, Meyers C Y:“Activities of Non—Classical Estrogens: Effects of (−)-, (+)-, and(±)-Z-Bisdehydrodoisynolic Acids In Vitro and on Body Weight in Male andFemale Rats.” The Endocrine Society, 80th Annual Meeting, Abstract(1998)), m.p. (recrystallized from acetone-ligroin) 204-205.5°C.(darkens) 204° C. The slightly off-white crystals rapidly deepen incolor in solution and more slowly in air. (+)-Z-BDDA and (−)-Z-BDDA wereprepared by the resolution of (±)-Z-BDDA-3-OMe through their respectiveisolated and purified L-menthyl esters according to the method reportedby Rometsch and Miescher. Rometsch R, Miescher K: “Die spaltung desracemates der n-bisdehydro-doisynolsäure. Über östrogene carbonsäurenX.” Helv Chim Acta 29:1231-1235 (1946). The crystalline (+)- and(−)-Z-BDDA so prepared exhibited a single TLC spot, and their ¹H- and¹³C-NMR spectra correctly identified their structure.

[0225] Experiment 1

[0226] Twenty-five male and 25 female Sprague-Dawley rats, 9-10 weeks ofage, were randomly assigned to groups of five animals, respectively, asa control group (no treatment) (C), for treatment with vehicle only (V),estradiol (E), (±)-Z-BDDA, or (+)-Z-BDDA. Each animal in group Vreceived a daily 0.1 cc injection of a 10% ethanol-90% olive oilsolution; the other treatments received their respective compound as adaily 0.1 cc injection, e.g., 2.5 μg of compound/g of body weight, in a10% ethanol-90% olive oil solution. A temperature of 21° C. and anartificial 12-h light-dark cycle was maintained in the animal room. Allanimals were maintained on standard chow in powdered form for fourweeks, then sacrificed after an overnight fast under i.p. pentobarbitalanesthesia (50 mg/kg). Animal weight and food intake were measuredweekly during the study, and the subsequent food-efficiency ratio [FER(gof weight change/g of food intake)] was determined. During sacrifice,blood was collected (via cardiac puncture) for cholesterol measurements.Immediately following sacrifice the fat pads and reproductive organswere removed and weighed.

[0227] Experiment 2

[0228] Twenty-five male and 25 female Wistar rats, 7-8 weeks of age,were randomly assigned to groups of five animals, respectively, fortreatment with vehicle only (V), estradiol (E), (±)-Z-BDDA, (+)-Z-BDDA,or (−)-Z-BDDA. Each animal in group V received a daily 0.1 cc injectionof a 10% ethanol-90% olive oil solution; the other treatments receivedtheir respective compound as a daily 0.1 cc injection, e.g., 2.5 μg ofcompound/g of body weight, in a 10% ethanol-90% olive oil solution. Atemperature of 21° C. and an artificial 12-h light-dark cycle weremaintained in the animal room. All animals were maintained on standardchow in powdered form, the males for five weeks and the females for sixweeks, then sacrificed after an overnight fast under i.p. pentobarbitalanesthesia (50 mg/kg). Animal weight and food intake were measuredweekly during the study and subsequent FER values were determined.During sacrifice, blood was collected (via cardiac puncture) forglucose, luteinizing hormone, prolactin and testosterone measurements.The amounts of luteinizing hormone, prolactin and testosterone weremeasured to help elucidate the target tissue of the respective compoundsin male and female rats. Immediately following sacrifice the fat padsand reproductive organs were removed and weighed.

[0229] Statistical Analyses

[0230] Experiments 1 and 2 employed a randomized design. All data wereanalyzed by one-way analysis of variance (ANOVA,)and post-hoccomparisons were made with Tukey pairwise comparisons test. Significancewas confirmed at p≦0.05 (SYSTAT 7.0, SPSS INC., 1997), and all valuesare reported as means±standard error of the mean.

Results

[0231] E2 and the three forms of Z-BDDA produced both similar anddistinct effects on reproductive parameters in male and female rats. Forexample, the results demonstrate that E2 and (+)-and (±)-Z-BDDA behavesimilarly in their effect on reproductive-organ weight: they promote anincrease in uterine weight and a decrease in testis weight, compared tothe control or vehicle alone (Table 1 and 2). Surprisingly, (−)-Z-BDDAdid not induce an increase (p<0.05) in uterine weight as observed with(+), (±)-Z-BDDA and estradiol (FIG. 1) but, like them, (−)-Z-BDDA,compared to the vehicle alone, caused weight reduction (p<0.05) of thetestis and prostate (Tables 1 and 2). TABLE 1 Experiment 1. The effectsof (+)-and (±)-Z-bisdehydrodoisynolic acid (Z-BDDA) and(+)-17β-estradiol on metabolic and reproductive parameters in male andfemale rats on treatment for 4 weeks* Cholesterol Uterus/testis ProstateTreatment Food intake (g) FER (food efficiency ratio) (mg/dl) Visceralfat (g) weight (g) weight (g) Female control 300.0 ± 28.6   0.079 ±0.010 91.6 ± 12.3 2.9 ± 0.3 0.42 ± 0.03 — vehicle^(†) 293.0 ± 26.1  0.040 ± 0.013 67.2 ± 12.5 1.6 ± 0.4¹ 0.66 ± 0.06 — estradiol^(¶) 371.6± 17.8   0.035 ± 0.011 44.9 ± 15.8¹ 0.8 ± 0.1¹ 1.58 ± 0.15 —(±)-Z-BDDA^(¶) 318.2 ± 21.8 −0.023 ± 0.027¹ 30.1 ± 2.8¹ 1.3 ± 0.1 2.04 ±0.45¹ — (+)-Z-BDDA^(¶) 315.2 ± 18.4   0.045 ± 0.017⁴ 35.1 ± 8.6¹ 2.1 ±0.3^(1,3) 2.57 ± 0.79^(1,2) — Male control 402.8 ± 25.0   0.134 ± 0.01382.1 ± 6.1 5.4 ± 0.7 3.82 ± 0.19 0.64 ± 0.10 vehicle^(†) 339.8 ± 22.6  0.087 ± 0.011 81.5 ± 3.9 8.2 ± 0.7¹ 3.55 ± 0.11 0.56 ± 0.03estradiol^(¶) 298.2 ± 18.8¹ −0.057 ± 0.012^(1,2) 51.5 ± 9.5^(1,2) 4.2 ±0.4² 0.94 ± 0.13^(1,2) 0.09 ± 0.01^(1,2) (±)-Z-BDDA^(¶) 344.6 ± 15.5−0.099 ± 0.009^(1,2) 36.2 ± 2.6^(1,2) 4.5 ± 0.4² 0.72 ± 0.06^(1,2) 0.08± 0.02^(1,2) (+)-Z-BDDA^(¶) 326.0 ± 9.5¹ −0.174 ± 0.027^(1,2,3,4) 24.6 ±2.2^(1,2,3) 5.1 ± 0.2² 0.84 ± 0.06^(1,2) 0.13 ± 0.04^(1,2,3)

[0232] TABLE 2 Experiment 2. The effects of (−)-, (+)-, and(±)-Z-bisdehydrodoisynolic acids (Z-BDDA) and (+)-17β-estradiol onmetabolic and reproductive parameters in male and female rats ontreatment for 5-6 weeks* Blood glucose Uterus/testis Prostate TreatmentFood intake (g) FER (food efficiency ratio) (mg/dl) weight (g) weight(g) Female vehicle^(†) 625.8 ± 49.3   0.071 ± 0.007 107.00 ± 11.47 1.22± 0.21 — estradiol^(¶) 699.6 ± 33.7   0.039 ± 0.004 100.80 ± 10.22 4.83± 1.30¹ — (±)-Z-BDDA^(¶) 704.0 ± 51.7   0.017 ± 0.003^(1,2)  80.40 ±5.66 4.25 ± 1.04¹ — (+)-Z-BDDA^(¶) 693.6 ± 72.3   0.026 ± 0.008¹  89.60± 6.15 4.02 ± 0.91¹ — (−)-Z-BDDA^(¶) 675.2 ± 37.9   0.011 ± 0.005^(1,2) 95.00 ± 8.33 1.44 ± 0.34^(2,3,4) — Male vehicle^(†) 525.8 ± 27.3   0.12 ± 0.02 134.80 ± 10.97 2.68 ± 0.26 0.25 ± 0.11 estradiol^(¶)590.4 ± 47.7    0.00 ± 0.01¹ 108.00 ± 10.90 0.74 ± 0.04¹ 0.06 ± 0.02(±)-Z-BDDA^(¶) 530.0 ± 33.0  −0.04 ± 0.01^(1,2)  98.60 ± 14.00 0.78 ±0.02¹ 0.16 ± 0.02 (+)-Z-BDDA^(¶) 676.0 ± 33.2  −0.02 ± 0.01¹  94.00 ±10.18¹ 0.61 ± 0.02¹ 0.02 ± 0.00¹ (−)-Z-BDDA^(¶) 650.0 ± 54.3  −0.01 ±0.01¹  88.00 ± 2.00¹ 0.57 ± 0.04¹ 0.03 ± 0.10¹

[0233] Parallel to their effects on reproductive parameters, E2 and thethree forms of Z-BDDA also elicited similar and distinct effects onmetabolic parameters in the male and female rats. For example, in thefemale rats in both experiments, (−)-and (±)-Z-BDDA appeared to repressweight gain slightly more than did (+)-Z-BDDA and E2, while in the malerats estradiol and the three Z-BDDA forms caused a dramatic reduction inbody weight (FIGS. 2 and 3).

[0234] Surprisingly, the specific enantiomers of Z-BDDA appeared toelicit a divergence between estrogenicity and weight repression in thefemale rats (FIGS. 1, 2, and 3 ), whereas in the male rats, all threeZ-BDDA forms not only elicited estrogenic-like effects (Table 1 and 2),but appeared to be more potent with respect to weight repression (FIGS.2 and 3). The male rats exhibited a reduction in visceral fat weightwhen treated with E2 and each of the three Z-BDDA forms, respectively,versus vehicle (Table 1). It is apparent from other metabolic parameters(i.e., food intake and FER) that the weight repression in weight-gainand even the weight reduction were independent of food intake (Table 1and 2). In fact, almost all of the difference in weight can be accountedfor by a decrease in food efficiency (Table 1 and 2).

[0235] In addition to the gross physiological parameters, metabolicprocesses were also examined. The female rats receiving (+)-Z-BDDA,(±)-Z-BDDA, or E2 exhibited a decrease in total cholesterol versuscontrol (Table 1). The males exhibited a more pronouncedcholesterol-lowering pattern (p<0.05), (±)-Z-BDDA having the mostprofound effect (Table 1). Furthermore, in the second experiment, malerats receiving either (−)-or (+)-Z-BDDA compounds exhibited a reduction(p<0.05) in blood glucose, while the (±)-Z-BDDA treated males and thefemales treated with all the Z-BDDA compounds demonstrated a trend(p=0.06) toward a reduction in blood glucose (Table 2).

[0236] In the same experiment, luteinizing hormone, prolactin, andtestosterone were measured to detect possible endocrine disruptioncaused by the Z-BDDA compounds. No significant changes in luteinizinghormone and prolactin were observed. However, compared to the vehicle,(±)-Z-BDDA as well as E2 caused a significant (p<0.05) testosteronesuppression in the male rats, a trend which was less pronounced with(−)- and (±)-Z-BDDA.

Conclusions

[0237] These results with the Z-BDDA compounds demonstrate that specificenantiomers of Z-BDDA appear to confer cardioprotective benefits (i.e.,reduction in cholesterol, body weight, blood glucose, and positivealterations in distribution of visceral fat). Wilson P W: “The Impact ofEstrogen on Cardiovascular Disease.” Perspective Studies: The FraminghamStudy. Postgrad Med 51-53:89-90 (1989); Cooper R L, Kavlock R J:“Endocrine Disruptors and Reproductive Development: A Weight-of-EvidenceOverview.” J Endocrinol 152:159-166 (1997); and Reubinoff B E, WurtmanJ, Rojansky N. Adler D, Stein P, Schenker J G, BrZeZinski A: “Effects ofHormone Replacement Therapy on Weight, Body Composition, FatDistribution, and Food Intake in Early Postmenopausal Women: AProspective Study.” Fertil Steril 64:963-968 (1995). The (−) enantiomerappears to minimize undesirable estrogenic effects on reproductivetissues. The Z-BDDA compounds exhibited a cholesterol-lowering effectconsistent with that elicited by other estrogenic compounds. See Heer J,Billeter J R, Miescher K: “Totalsynthese der racemischenbisdehydro-doisynolsäure. Über oestrogene carbosäuren IV.” Helv. Chim.Acta 28:1342-1354 (1945); Ke H Z, Chen H A, Simmons H A, Qi H, CrawfordD T, Pirie C M, Chidsey-Frink K L, Ma Y F, Jee W S S, Thompson D D:“Comparative Effects of Droloxifene, Tamoxifen, and Estrogen on Bone,Serum Cholesterol, and Uterine Histology in the Ovariectomized RatModel.” Bone 20:31-39 (1997); Sato M, Rippy M K, Bryant H U:“Raloxifene, Tamoxifen, Nafoxidine, or Estrogen Effects on Reproductiveand Nonreproductive Tissues in Ovariectomized Rats.” FASEB J 10:905-912(1996); Dodge J A, Glasebrook A L, Magee D A, Phillips D L, Sato M,Short LL, Bryant H U: “Environmental Estrogens: Effects on CholesterolLowering and Bone in the Ovariectomized Rat.” J Steroid Biochem MolecBiol 59:155-161(1996); and Hart J E: “Endocrine Pathology of Estrogens:Species Differences.” Pharmac Ther 47:203-218 (1990). Surprisingly, thehypocholesterolemic, weight-repressing, and visceral fat-reducingeffects were demonstrated in reproductively intact male and female rats.This effect may be unique among non-steroidal estrogens. For example,raloxifene has no significant clinical effects in healthy, menstruatingwomen. Heywood R, Wadsworth P F: “The Experimental Toxicology ofEstrogens.” Pharmac Ther 8:125-142 (1980). While raloxifene as well astamoxifen and nafoxidine seem to elicit a cholesterol-lowering and aminimal weight-repressing effect in ovariectomized animals, the Z-BDDAcompounds appear to be much more effective and, moreover, produce thiseffect in reproductively intact animals. Meyers C Y, Lutfi H G, Adler S:“Transcriptional Regulation of Estrogen-Responsive Genes byNon-Steroidal Estrogens: Doisynolic and Allenolic acids.” J SteroidBiochem Molec Biol 62:477-489 (1997); Heer J, Billeter J R, Miescher K:“Totalsynthese der racemischen bisdehydro-doisynolsäure. Über oestrogenecarbosäuren IV.” Helv. Chim. Acta 28:1342-1354 (1945); Ke H Z, Chen H A,Simmons H A, Qi H, Crawford D T, Pirie C M, Chidsey-Frink K L, Ma Y F,Jee W S S, Thompson D D: “Comparative Effects of Droloxifene, Tamoxifen,and Estrogen on Bone, Serum Cholesterol, and Uterine Histology in theOvariectomized Rat Model.” Bone 20:31-39 (1997); Sato M, Rippy M K,Bryant H U: “Raloxifene, Tamoxifen, Nafoxidine, or Estrogen Effects onReproductive and Nonreproductive Tissues in Ovariectomized Rats.” FASEBJ 10:905-912 (1996); and Heywood R, Wadsworth P F: “The ExperimentalToxicology of Estrogens.” Pharmac Ther 8:125-142 (1980). Being observedin intact, non-castrate male and female animals, these effects suggestclinical applications for these or similar compounds in treating pre- aswell as post-menopausal women, and males at risk for cardiovascular andprostatic disease.

[0238] The distinct effects elicited by all three Z-BDDA forms on bodyweight, food intake, FER, and visceral fat appear to becompound-specific and somewhat divergent from the effects elicited byE2. It is apparent from the food intake and FER data that the repressionin body-weight gain was independent of the quantity of food consumed.The fact that almost all of the variation in weight can be accounted forby a decrease in food efficiency points to a metabolic alterationelicited by the Z-BDDA compounds rather than appetite suppression as theweight-repressing mechanism. This finding is in contrast to the effectsof other estrogenic compounds on body weight. While some other estrogensmay cause weight repression, in those cases it appears to be compound-,species-, and gender-specific and, in sharp contrast to the resultsobtained with the present BDDA compounds, can be explained by areduction in food intake. In further contrast to the present resultswith the BDDA compounds, these effects elicited by other estrogens arereported in studies with castrated rather than reproductively intactanimals. See Heer J, Billeter J R, Miescher K: “Totalsynthese derracemischen bisdehydro-doisynolsäure. Über oestrogene carbosäuren IV.”Helv. Chim. Acta 28:1342-1354 (1945); Ke H Z, Chen H A, Simmons H A, QiH, Crawford D T, Pirie C M, Chidsey-Frink K L, Ma Y F, Jee W S S,Thompson D D: “Comparative Effects of Droloxifene, Tamoxifen, andEstrogen on Bone, Serum Cholesterol, and Uterine Histology in theOvariectomized Rat Model.” Bone 20:31-39 (1997); Sato M, Rippy M K,Bryant H U: “Raloxifene, Tamoxifen, Nafoxidine, or Estrogen Effects onReproductive and Nonreproductive Tissues in Ovariectomized Rats.” FASEBJ 10:905-912 (1996); Dodge J A, Glasebrook A L, Magee D A, Phillips D L,Sato M, Short L L, Bryant H U: “Environmental Estrogens: Effects onCholesterol Lowering and Bone in the Ovariectomized Rat.” J SteroidBiochem Molec Biol 59:155-161 (1996); and Hart J E: “Endocrine Pathologyof Estrogens: Species Differences.” Pharmac Ther 47:203-218 (1990).

[0239] These results also suggest that (−)-Z-BDDA appears to exhibitboth estrogenic and anti-estrogenic activities in female rats. This wasnot the case for the males, and may be dependent on the interaction of(−)-Z-BDDA with endogenous E2. Racemic Z-BDDA and its two enantiomers,while all promoting weight-repressing effects in female rats, differedin their capacity to elicit uterotropism, a classic assay for estrogenicactivity. Surprisingly, (−)-Z-BDDA did not induce the significantincreases in uterine weight observed with (+)- or (±)-Z-BDDA or E2. Incontrast to the results observed in the chronic treatment study, it haspreviously been demonstrated that the (−) enantiomer of (±)-Z-BDDA isthe enantiomer responsible for the high uterotropic activity observedwhen administered acutely. Anner G, Miescher K: Hydrierungs-UndUmlagerungs-Reaktion in der Doisynolsäure-Reihe. Oestrogene CarbonsäurenXII. Helv. Chim. Acta 29 (1946) 1889-1895; Die totalsyntheses vonracemischen doisynolsäuren XXI. Über oestrogene carbonsäueren. ibid30:1422-1432 (1947); Tschopp E: “Wirksamkeit, organconzentration undausscheidung der 7-methyl-bisdehydro-doisynolsäure.” Helv PhysiolPharmacol Acta 4:401-410 (1946); Tschopp E: “Die oestrogene wirkung derbisdehydrodoisynolsäure und ihre derivate.” Helv Physiol Pharmacol Acta4:271-284 (1946); Rometsch R, Miescher K: “Die spaltung des racematesder n-bisdehydro-doisynolsäure. Über östrogene carbonsaiuren X.” HelvChim Acta 29:1231-1235 (1946); and Terenius L: “Differential InhibitionIn Vitro of 17β-Estradiol Binding in the Mouse Uterus and Vagina byOptical Antipodes of Estrogen.” Molec Pharmac 4:301-310 (1968). Thebasis of the difference between the results presented herein and thosereported previously is not known. However, in addition to differences induration of treatment, other factors that may have contributed to thelack of uterotropism elicited by (−)-Z-BDDA in the present studiesinclude the dosages used and the species and ages of the animals.

[0240] Of further interest are the differences observed in the potencyof the Z-BDDA compounds when the in vivo results are compared witheither cell-culture assays measuring activation of estrogen receptor, orwith in vitro assays of relative receptor-binding affinity. Numerouscompetitive binding-inhibition studies with the classical estrogenreceptors (ERα) have demonstrated that the binding affinity of(±)-Z-BDDA is much lower than that of estradiol. Meyers C Y, Kolb V M,Gass G H, Rao B R, Roos C F, Dandliker W B: “Doisynolic-TypeAcids—Uterotropically Potent Estrogens which Compete Poorly withEstradiol for Cytosolic Estradiol Receptors. J Steroid Biochem31:393-404 (1988); Soto A M, Meyers C Y, Sonnenschein C: “How Many RingsCan be Cleaved from a Steroidal Estrogen While Preserving its EstrogenicActivity?“The Endocrine Society, 70th Annual Meeting, Abstract (1988);and Banz J, Winters T A, Hou Y, Adler S, Meyers C Y: “Activities ofNon—Classical Estrogens: Effects of (−)-, (±)-, and(±)-Z-Bisdehydrodoisynolic Acids In Vitro and on Body Weight in Male andFemale Rats.” The Endocrine Society, 80th Annual Meeting, Abstract(1998). These results were substantiated recently by direct bindingstudies using preparations of human ERα, and are in agreement withprevious results with (−)-Z-BDDA, which were determined with mouseuterine tissue in competitive binding-inhibition studies. Terenius L:“Differential Inhibition In Vitro of 17β-Estradiol Binding in the MouseUterus and Vagina by Optical Antipodes of Estrogen.” Molec Pharmac4:301-310 (1968); and Meyers C Y, Lutfi HG, Adler S: “TranscriptionalRegulation of Estrogen-Responsive Genes by Non-Steroidal Estrogens:Doisynolic and Allenolic acids.” J Steroid Biochem Molec Biol 62:477-489(1997). Hence, there is an apparent activity/binding paradox, suggestingthat the classic estrogen receptor, ERα, may not be the exclusivereceptor or pathway mediating the actions of Z-BDDA compounds, orpossibly even those of estradiol. Meyers C Y, Kolb V M, Gass G H, Rao BR, Roos C F, Dandliker W B: “Doisynolic-Type Acids—UterotropicallyPotent Estrogens which Compete Poorly with Estradiol for CytosolicEstradiol Receptors. J Steroid Biochem 31:393-404 (1988).

[0241] Recently, a new form of estrogen receptor, ERA, has beenidentified, and its role in estrogenic regulation in various targettissues and its affinity for non-steroidal ligands are currently beingdefined. Kuiper G G, Carlsson B, Grandien K, Enmark E, Haggblad J,Nilsson S, Gustafsson J: “Comparison of the Ligand Binding Specificityand Transcript Tissue Distribution of Estrogen Receptors α and β.”Endocrinology 138:863-870 (1997); and Pace P, Taylor J, SuntharalingamS, Coombes R C, Ali S: “Human Estrogen Receptor β Binds DNA in a MannerSimilar to and Dimerizes with Estrogen Receptor α.” J Biol Chem272:25832-25838 (1997). Initial studies comparing the classical ERα andthe novel estrogen receptor ERβ show very similar results. The bindingaffinity of (+)-Z-BDDA is even lower than that of the (−) enantiomer,and both enantiomers have a much lower affinity for estrogen receptorsthan does estradiol, whether measured via direct receptor binding assaysor by generating dose-response profiles using activation ofestrogen-responsive reporter genes in cell-culture systems. Banz J,Winters TA, Hou Y, Adler S, Meyers C Y: “Activities of Non—ClassicalEstrogens: Effects of (−)-, (+)-, and (±)-Z-Bisdehydrodoisynolic AcidsIn Vitro and on Body Weight in Male and Female Rats.” The EndocrineSociety, 80th Annual Meeting, Abstract (1998). The evaluation of ERβ hasnot resolved the apparent paradox. However, the use of heterodimers ofERα and ERβ has not been evaluated, and may add a further degree ofcomplexity to this binding/activity evaluation.

[0242] Alternatively, there is evidence that in vivo, serum-bindingproteins could account for part of the activity/binding paradoxemanating from a comparison of estradiol on one hand and the threeZ-BDDA forms on the other. DanZo B J: “Environmental Xenobiotics MayDisrupt Normal Endocrine Function by Interfering with the Binding ofPhysiological Ligands to Steroid Receptors and Binding Proteins.”Environ Health Perspect 105:294-301 (1997); and Nagel S C, vom Saal F S,Thayer K A, Dhar M G, Boechler M, Welshons W V: “Relative BindingAffinity—Serum Modified Access Assay Predicts the Relative In VivoBioactivity of the Xenoestrogens Bisphenol A and Octylphenol.” EnvironHealth Perspect 105:70-76 (1997). Steroid-hormone binding globulin(SHBG) and serum albumin appear to have a much higher affinity forestradiol than for many environmental and synthetic estrogens. Thus, invivo, there is a relatively higher level of free versus bound compoundcompared to estradiol than would be predicted from in vitro bindingstudies alone. In addition, nonsteroidal environ-mental and syntheticestrogens may also elicit biological effects independent of theligand-estrogen receptor complex (i.e., antioxidant and enzymemodulation). Wehling M: “Specific, Nongenomic Actions of SteroidHormones.” Annu Rev Physiol 59:365-393 (1997); Akiyama T, Ishida J,Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y:“Genistein, A Specific Inhibitor of Tyrosine—Specific Protein Kinases.”J Biol Chem 262:5592-5595 (1987); Peterson G, Barnes S: “GenisteinInhibits Both Estrogen and Growth Factor—Stimulated Proliferation ofHuman Breast Cancer Cells. Cell Growth & Differentiation 7:1345-1351(1996); Spink D C, Johnson J A, Connor S P, Aldous K M, Gierthy J F:“Stimulation of 17 Beta-Estradiol Metabolism in MC F-7 Cells byBromochloro-and Chloromethyl—Substituted DibenZo-p-dioxins andDibenzofurans: Correlations with Antiestrogenic Activity.” Journal ofToxicology & Environmental Health 41:451-466 (1994); Behl C, Skutella T,LeZoualch F, Post A, Widmann M, Newton C J, Holsboer F: “NeuroprotectionAgainst Oxidative Stress by Estrogens: Structure-Activity Relationship.”Mol Pharmacol 51:535-541 (1997); Wiseman H, O'Reilly J: “Oestrogens asAntioxidant Cardioprotectants.” Biochemical Society Transactions25:54-59 (1997); and Smith C L, Conneely O M, O'Malley B W: “Modulationof the Ligand-Independent Activation of the Human Estrogen Receptor byHormone and Antihormone.” Proc Natl Acad Sci 90:6120-6124 (1993). Thefinal in vivo effect of these compounds may reflect all of thesecontributions.

[0243] The foregoing data, generated in intact, non-castrated male andfemale animals, indicate that the observed effects, unlike thosereported in comparable studies with tamoxifen, nafoxidine, orraloxifene, are not obscured by endogenous estradiol. Heer J, Billeter JR, Miescher K: “Totalsynthese der racemischen bisdehydro-doisynolsäure.Über oestrogene carbosäuren IV.” Helv. Chim. Acta 28:1342-1354 (1945);Ke H Z, Chen H A, Simmons H A, Qi H, Crawford D T, Pirie C M,Chidsey-Frink K L, Ma Y F, Jee W S S, Thompson D D: “Comparative Effectsof Droloxifene, Tamoxifen, and Estrogen on Bone, Serum Cholesterol, andUterine Histology in the Ovariectomized Rat Model.” Bone 20:31-39(1997); Sato M, Rippy M K, Bryant H U: “Raloxifene, Tamoxifen,Nafoxidine, or Estrogen Effects on Reproductive and NonreproductiveTissues in Ovariectomized Rats.” FASEB J 10:905-912 (1996); and HeywoodR, Wadsworth P F: “The Experimental Toxicology of Estrogens.” PharmacTher 8:125-142 (1980). The Z-BDDA compounds cause weightrepression/reduction in male and female rats via an unknown mechanism.The results demonstrate remarkable selective estrogen receptor modulator(SERM) activity, and strongly suggest clinical applications for thesecompounds in peri- as well as post-menopausal women. Furthermore, theysuggest clinical applications for these compounds (or appropriatederivatives thereof) in males at risk for cardiovascular and prostaticdisease.

Example 3 Effects of Z-Bisdehydrodoisynolic Acids on In situ Apoptosisin Primary Porcine Granulosa Cells

[0244] Estrogens have been found to decrease ovarian follicle atresia(Tilly et al. (1991) Endocrinology 129:2799-2801), which in turn couldincrease the number of follicles recruited and thus ovulated eachmenstrual or estrous cycle in humans and animals, respectively.Apoptosis, or programmed cell death, is the underlying mechanism forfollicular atresia. This experiment was performed to determine if BDDAsaffect follicular cell apoptosis.

[0245] Materials and Methods

[0246] Tissue Culture

[0247] Porcine ovaries were obtained from local packing plants andtransported to the laboratory on ice-cold Hank's balanced salt solution(HBSS). Each of the follicles was aspirated with an insulin syringe, andthe follicular fluid was centrifuged at 3000 rpm and 4° C. for 15 min.The supernatant was poured off and the cells were washed in 5 mls ofcold HBSS and centrifuged for another 10 min. The cells were againresuspended in HBSS, and the number of viable cells counted under themicroscope using a hematocytometer. Once the number of cells wasdetermined, the cells were centrifuged again for 10 min. and resuspendedin the appropriate volume of Eagle's minimum essential media (MEM)containing 10% fetal bovine serum (FBS) and antibiotic/antimycotics. Thecells were plated at 250,000 per well in 8 chamber microscope slides(Nunc, Naperville, Ill.) which were pre-treated with poly-L-lysine for10 min. The slides were incubated at 5% CO₂/95% air at 37° C..Approximately 12 hours later, the medium was removed by vacuum andreplaced with pre-warmed, serum-free MEM. Cells were then treated for2-3 hrs with MEM only to wash out any estrogen effects from the serum(Winters et al. (1994) Biol. Reprod. 50 (Suppl. 1):113; Suttner et al.(1998) Biol. Reprod. 59 (Suppl.): (Accepted for publication).

[0248] Treatments

[0249] Following serum-free MEM treatment, cells were treated for 24 hrwith (+) or (−) enantiomers of Z-BDDA or estradiol at 0.1, 1, and 10 μM,or EtOH vehicle control in serum-free MEM.

[0250] Apoptosis Assay

[0251] Cells were subsequently processed using an in situ apoptosisassay kit (Apotag-Plus In Situ Apoptosis Kit—Peroxidase, Edition 1.1.,Oncor, Gaithersburg, Md., 1995) to study ovarian apoptosis (Suttner etal., 1998). The slides were washed in two changes of PBS for 5 min each,and then quenched in 2% hydrogen peroxide in methanol for 5 min. at roomtemperature. After pre-treatment with an equilibration buffer, 13 ul ofTerminal deoxynucleotidyl Transferase (TdT) diluted with reaction bufferand distilled water were added, and the slides were incubated for 1 h at37° C. in a humidified chamber. After this 1 h period, the slides wereput in pre-warmed stop wash buffer in the incubator for 30 min to stopthe reaction. Next, the slides were washed in three changes of PBS for 5min. each, and the anti-digoxigenin peroxidase was placed on the slidesfor 30 min. in a humidified chamber at room temperature. Once this timewas up, the slides were washed in four changes of PBS for 5 min. eachand stained with diaminobenzidine substrate solution for 15-20 min. Thisyielded a brown stain in apoptotic cells. After washing in three changesof distilled water for 1 min., followed by a 5 min. wash, the slideswere counterstained in methyl green for 8 min. This yielded a blue/greenstain in non-apoptotic cells. Once counterstained, the specimens werewashed in three changes of distilled water and 100% butanol,respectively, by dipping 10 times in the first and second washes,followed by 30 sec. in the third wash. The slides were cleared in threewashes of xylene for 2 min. each and then mounted under coverslips withpermount.

[0252] Image Analysis

[0253] The degree of apoptosis for the colorimetric apoptosis assay wasquantified microscopically using an image analysis system (Optimas 5.23,Optimas Users Guide, 5th Edition, Redmond, Wash.). Ten measurements(captured images) were taken for each concentration based on apre-determined grid. Brown and blue/green color thresholds for apoptoticand non-apoptotic cells, respectively, were set for each captured image.Percentage area of each color was then quantitated using the imageanalysis system. Data were then transferred to a spreadsheet (Excel,Microsoft Corp., Redmond, Wash.) for sorting before statisticalanalysis. This procedure was repeated for each slide and eachconcentration in duplicate.

[0254] Statistics

[0255] Statistical analysis was performed using a statistical program(SAS, 1988, SAS/STAT User's Guide. Statistical Analysis Institute, Cary,N.C.). Contrast analyses were run for the weekly experiments, and all ofthe treatments were compared. The level of significance was determinedat p<0.05.

Results

[0256] (−)-Z-BDDA treatment decreased (P<0.01) mean apoptosis (%are^(a)) from 63.4% in the controls to 26.1% in treated cells.(+)-Z-BDDA treatment did not appreciably change mean apoptosis in thecontrols (68.4%) vs. the treated cells (61.1%). Estradiol treatmentcombined was not different from controls; however, percent apoptosis waslower (P<0.05) at 10 μM estradiol (23.8%). In addition, (−)-Z-BDDAtended to decrease (P=0.06) percent apoptosis vs. estradiol (46.5).

Conclusions

[0257] These results indicate that (−)-Z-BDDA has the ability todecrease apoptosis in granulosa cells from the ovarian follicle of aporcine experimental model. Decreased follicle apoptosis could lead tomore follicle recruitment and ovulations in the mammalian ovary. Theinhibition of granulosa cell apoptosis by (−)-Z-BDDA appears to be moresubstantial than that of estradiol. The (+)-enantiomer did not appear tohave an effect in these experiments. However, (+)-Z-BDDA could be activeat a higher concentration, or be acting as an antiestrogen inhibitingthe estrogenic effect seen with (−)-Z-BDDA and estradiol. These resultssuggest that the use of (−)-Z-BDDA in human and/or veterinary medicinecould lead to more follicle recruitment and ovulations, thus increasingfertility. (+)Z-BDDA may may have applications as a birth control drug.In addition, the BDDAs could potentially be used to modulate otherphysiological processes controlled by apoptosis, including maturation ofthe immune system, embryonic development, luteolysis, male patternbaldness, cancer, tissues responding to thermal and metabolic stress,tissues responding to hormonal stimuli (especially estrogens), andnormal tissue turnover (Bowen et al. (1990) Programmed Cell Death inTumors and Tissues, Chapman & Hall, New York, N.Y.).

Example 4 Differential Effects of Estrogenic Carboxylic Acids on theProstate and Testis of Male Rats

[0258] Estrogens have been used in the treatment of prostate cancer;however, these estrogens have negative feminizing side effects. Theseinclude shrinkage of the testis and accessory glands (including theprostate), gynecomastia, salt and water retention, and inhibition ofother secondary male sex characteristics (including loss of libido andimpotence). Gudziak, M R, and A Y Smith. “Hormonal Therapy for Stage DCancer of the Prostate” West J Med 160:351-359 (1994). In addition,estrogen therapy in males leads to a three-fold increased risk ofthromboembolic events (including heart attacks, strokes, and bloodclots). Glashan, R W, and M R G Robinson. “Cardiovascular Complicationsin the Treatment of Prostatic Carcinoma.” Br J Urol 53:624-627 (1981).Since estrogen treatment in males causes these undesirable effects,estrogens are only used in severe prostate carcinoma, and are notusually used in other prostatic conditions such as benign prostatehypertrophy. Jacobi, G H. “Hormonal Treatment of Metastatic Carcinoma.”In: The Prostate, pp. 119-128.(J M Fitzpatrick and R J Krane, eds.,Churchill Livingstone, New York, N.Y. 1989); de Klerk, D P, and F Allen.“Medical Therapy for Benign Prostatic Hyperplasia.” In: The Prostate,pp. 119-128 (J M FitZpatrick and R J Krane, eds., Churchill Livingstone,New York, N.Y. 1989).

[0259] This study was undertaken to determine the effects thatenantiomers of the estrogenic carboxylic acids, Z-bisdehydrodoisynolicacids (BDDA) and hydroxyallenolic acids (HAA), have on the prostate,testis, and other physiological parameters in male rats. As reportedbelow, the results demonstrate that these compounds possess utility as atherapy for prostatic disease, as well as in other clinical applicationsin males.

[0260] Materials and Methods Sixty male Sprague-Dawley rats, 7-8 weeksof age, were randomly assigned to groups of ten animals. Each group wasrandomly assigned to one of six treatments: Vehicle control (C),Estradiol-17β (E), (−)-Z-BDDA, (+)-Z-BDDA, (−)-HAA, and (+)-HAA. Thecompounds were all administered at a dose of 0.1 μg/g of body weight in0.1 cc once a day for 6 weeks. The estrogenic compounds were dissolvedin 10% ethanol and 90% olive oil vehicle. A temperature of 21° C. and anartificial 12 h light-dark cycle were maintained in the animal room. Allanimals were maintained on standard chow in powdered form for six weeks,and then sacrificed after an overnight fast under i.p. pentobarbitolanesthesia (50 mg/kg). Animal weight was measured weekly during thestudy. During sacrifice, blood was collected via cardiac puncture.Immediately following sacrifice, the fat pads, livers, pituitaries,testes, seminal vesicles, and prostate were removed and weighed, andsnap frozen in liquid nitrogen. Prostates and one testis from 2-3animals in each treatment group were fixed in 10% formalin forhistological examination. These tissues were fixed overnight, blocked inparaffin, sectioned at 4 μm, stained with Hematoxylin and Eosin, coverslipped, and examined microscopically. Quantitative results weresubjected to an analysis of variance and means separated by a Tukey'sTest (SYSTAT, Chicago, Ill.).

[0261] Results

[0262] Rats in all five estrogen treatments showed a significantdecrease (P<0.05) in weight gain compared to that in rats in the control(C) group (Table 3). The (+)-Z-BDDA-treated rats gained more (P<0.05)weight than the estradiol-treated, (−)-Z-BDDA-treated, (−)-HAA-treated,and (+)-HAA-treated rats. The (−)-Z-BDDA-treated rats had the lowestweight change, and was lower (P<0.05) than that in the control,estradiol-treated, (+)-Z-BDDA-treated, and (+)-HAA-treated groups.

[0263] Prostate weights as a percentage of bodyweight were lower(P<0.05) than that in controls in all five estrogen treatments (Table3). The weights of testes and seminal vesicles as a percentage ofbodyweight were lower (P<0.05) than that of control rats in theestradiol-treated, (−)-Z-BDDA-treated, (−)-HAA-treated, and(+)-HAA-treated rats (Table 1). The (+)-Z-BDDA-treated rats did not havesignificantly smaller testes or seminal vesicles as a percentage ofbodyweight, although gross testes weights unadjusted for bodyweight werelighter (P<0.05) than those in control rats (data not shown). There wereno obvious signs of gynecomastia in any of the rats. TABLE 3 The effectsof (−)- and (+)-Z-bisdehydrodoisynolic acids (BDDA), (−)- and(+)-hydroxyallenolic acid (HAA), and (+)-17β-estradiol (E) on metabolicand reproductive parameters in male rats on treatment for 6 weeks*Testis Weight as % Prostate Weight as % Seminal Vesicle Weight as %Treatment Body Weight (g) Body Weight Body Weight Body Weightvehicle^(†) 336.3 ± 4.9 1.14 ± 0.05 0.16 ± 0.02 0.21 ± 0.03 E^(¶) 207.2± 4.6¹ 0.49 ± 0.13^(1,4) 0.05 ± 0.00¹ 0.02 ± 0.00¹ (−)-BDDA^(¶) 166.5 ±4.5^(1,2,4) 0.31 ± 0.02^(1,4) 0.08 ± 0.00¹ 0.06 ± 0.02⁴ (+)-BDDA^(¶)234.8 ± 10.1^(1,2) 0.98 ± 0.15 0.05 ± 0.01¹ 0.23 ± 0.12² (−)-HAA^(¶)180.9 ± 4.5^(1,2,4) 0.31 ± 0.01^(1,4) 0.08 ± 0.01¹ 0.04 ± 0.00^(1,4)(+)-HAA^(¶) 196.2 ± 5.2^(1,4,5) 0.38 ± 0.02^(1,4) 0.08 ± 0.01¹ 0.10 ±0.04

[0264] Histological examination of the prostate showed normal alveoli inthe control and (+)-Z-BDDA-treated rats, with the tubules and alveolibeing slightly smaller only in the (+)-Z-BDDA-treated rats (FIGS. 4a-f).However, the alveoli showed significant degrees of atrophy in the otherfour treatments, with the (−)-HAA-treated rats displaying the largestdegree of atrophy. In the testis, spernatogenesis and Leydig cells werenormal in the control and (+)-Z-BDDA-treated rats, but were severelyattenuated in rats in the other four groups (FIGS. 5a-f). Rats in thesefour treatment groups had spermatogenesis halted in late meiosis, earlyspermiogenesis. The estradiol-treated rats showed spermatogenesis haltedat round (Golgi phase) spermatids, and Leydig cells were small. The(−)-Z-BDDA-treated rats were halted primarily at the secondaryspermatocyte phase, with a few spermatogenic cells reaching roundspermatid. The (−)-Z-BDDA-treated rats also had severely atrophiedLeydig cells, the smallest of all the treatments. The (−)-HAA-treatedrats were also halted at round spermatid, with a few reaching cap phase.The (+)-HAA-treated rats were halted at round spermatid, with a fewspermatogenic cells showing elongation (acrosome phase). BothHAA-treated groups had smaller Leydig cells than control and(+)-Z-BDDA-treated rats.

Conclusions

[0265] These results demonstrate that the estrogenic carboxylic acidsBDDA and HAA significantly reduce the size of the prostate inpost-pubertal male rats, and suggests their use in the treatment ofprostatic disease. This phenomenon may occur via an estrogen-inducedapoptotic mechanism. Treatment with the (+)-Z-BDDA enantiomer resultedin a different effect from that observed with the other estrogeniccompounds in that testis size, and more importantly spermatogenesis andLeydig cell function, was not compromised. The other estrogens used inthis study significantly shrank the testes, and decreased its gameticand endocrine function. As in the testes, (+)-Z-BDDA also did notsignificantly shrink the seminal vesicles. The observation that(+)-Z-BDDA shrinks the prostate without appreciably affecting the testesor seminal vesicles is novel among estrogenic compounds, and may beindicative of selective estrogen receptor modulation (SERM) activity inmales. SERM activity has been reported in the female, with compoundssuch as tamoxifen, nafoxidine, and raloxifene, but not in males. Thisdifferential effect of (+)-Z-BDDA also appears to be dependent on dose,since previous studies have shown that a dose 25 times higher (2.5 μg/gbodyweight) shrank the testis, similar to the effect of estradiol and(−)-Z-BDDA. Note Example 2, above, and Banz, W J, T A Winters, Y-Q Hou,S R Adler, and C Y Meyers. “Comparative Effects of the SelectiveEstrogen Receptor Modulators (−)-, (+)-, and (±)-Z-BisdehydrodoisynolicAcids on Metabolic and Reproductive Parameters in Male and Female Rats.”Horm Metab Res 30:730-736 (1998).

[0266] Since the effects of the BDDA and HAA estrogenic carboxylic acidswere observed in intact, non-castrate male rats, the present datasuggest clinical applications for these or similar compounds in treatingmales with prostatic disease. These applications could be alone or incombination with other treatments or therapies. The (−)-Z-BDDA and bothHAA enantiomers appear to be useful in treating severe prostaticcarcinoma since they cause atrophy of the prostate, and probablydecrease the androgen secretion of the testis, which is indicative ofthe atrophy of the Leydig cells. Androgens exacerbate the division andmetastasis of prostatic cancer cells. Gudziak, M R, and A Y Smith.“Hormonal Therapy for Stage D Cancer of the Prostate” West J Med160:351-359 (1994). The BDDA compounds, and possibly the HAA compounds,may have advantages over other estrogen therapies in that they alsolower certain cardiovascular risk factors. Note Example 2, above, andBanz, W J, T A Winters, Y-Q Hou, S R Adler, and C Y Meyers. “ComparativeEffects of the Selective Estrogen Receptor Modulators (−)-, (+)-, and(±)-Z-Bisdehydrodoisynolic Acids on Metabolic and ReproductiveParameters in Male and Female Rats.” Horm Metab Res 30:730-736 (1998)..Other estrogen therapies have well-documented cardiovascular sideeffects. Jacobi, G H. “Hormonal Treatment of Metastatic Carcinoma.” In:The Prostate, pp.119-128.(J M Fitzpatrick and R J Krane, eds., ChurchillLivingstone, New York, N.Y. 1989). In addition to prostate cancer,(+)-Z-BDDA appears to have utility in the treatment of benign prostatehypertrophy (BPH) since the prostate is reduced without compromisingspermatogenesis and/or androgen production by the testes. In addition,even though (+)-Z-BDDA shrank the prostate, histological analysisindicates that the exocrine function of this accessory gland is notappreciably compromised. The exocrine function of the seminal vesicleswith (+)-Z-BDDA is probably also unaffected. Therefore, together with noeffect on spermatogenesis, semen production should not be affected.

[0267] Other applications of these and related estrogenic carboxylicacids suggested by the present data include treatment of otherandrogen-responsive physiological or pathological conditions, a methodof male birth control, and a means for chemical castration in males.

Example 5 Effects of Z-Bisdehydrodoisynolic Acids on AntioxidantCapacity in an Oxidized LDL Lag Time Assay

[0268] The effects of several synthetic and environmental estrogens,i.e., (+)-and (−)-Z-BDDA, (+)-hydroxyvallestril (allenolic acid) and(−)-hydroxyvallestril (allenolic acid), genistein (soy phytoestrogen),daidzein (soy phytoestrogen), 4-hydroxy-tamoxifen, and estradiol (E2),on antioxidant capacity in an oxidized LDL lag time assay were comparedin order to assess the antioxidant activity of these compounds.

[0269] Experiments were carried out on dialyzed LDL collected from fourfasted persons. The LDL was used within 10 days of dialysis. Theoxidizing agent was 3 μM Cu₂SO₄; phosphate buffered saline was used tocontrol pH, and all drugs were dissolved in ethanol; finalconcentrations of each drug in the assays were 10⁻⁴, 10⁻⁵, 10⁻⁶, and10⁻⁷ M. The combined results are shown in FIG. 6.

[0270] Relative to the LDL/Cu curve, the (+)- and (−)-Z-BDDA curves wereshifted to the right and somewhat flattened (data not shown). This shiftin lag time to the right and flattening of the curves indicates that theZ-BDDAs exhibited significant antioxidant activity. Similar effects werealso observed in the case of (+)-allenolic acid, (−)-allenolic acid, and4-hydroxytamoxifen. Less antioxidant activity was observed withgenistein and daidzein. Estradiol exhibited very little antioxidantactivity under these conditions (data not shown).

[0271] The results indicate that (+)- and (−)-Z-BDDA, (+)- and(−)-hydroxyvallestril, and 4-hydroxytamoxifen were the most potentantioxidants.

[0272] Taken together, the results presented in Examples 2-5 suggestthat the non-steroidal, estrogenically active carboxylic acids of thepresent invention can be used in efficacious treatment programs forendocrine- and non-endocrine responsive conditions in males and females,e.g., prostatic disease, hormone-responsive cancers, osteoporosis,therapeutic applications for pre- and post-menopausal women, Alzheimer'sdisease, male pattern baldness, and as fertility (anti-atresia) andanti-fertility agents. These results further suggest clinicalapplications for the compounds disclosed herein, as well as appropriatederivatives thereof, in males at risk for cardiovascular disease viadecreased oxidation of LDL, for reduction of cholesterol, blood glucose,and body weight, and to achieve positive alterations in body fatdistribution. These results also suggest methods for treating orpreventing prostatic diseases including benign prostate hyperplasia andother related conditions, androgen-responsive pathological conditions inmales, and methods for male birth control and chemical castration,employing estrogenic carboxylic acids.

One-Pot Asymmetric Synthesis of (+)- and(−)-3-[2-(6-Methoxynaphthyl)]-2,2-dimethylpentanoic Acid Esters

[0273] In addition to compounds, compositions, and methods for treatingdiseases, symptoms, and conditions responsive to the compounds disclosedherein, the present invention also provides new synthetic methods forpreparing certain of these compounds. In particular, the presentinvention provides a direct one-pot synthesis to produce esters of3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid (Scheme 4) fromcommercially available starting material. These esters can then beeasily hydrolyzed under basic or acidic conditions to give 2 or 3.Although there are three reaction steps in this synthetic route,separation of intermediates is unnecessary, lowering the cost ofproduction by saving chemicals and manpower, and increasing productyield.

[0274] When a chiral R* group is used (Scheme 4), an asymmetricinduction in the Michael addition step is expected. By using differentchiral R* groups, it is possible to obtain one or the other enantiomerdirectly from the reaction, eliminating the resolution step and furtherlowering the cost of production.

[0275] This synthetic scheme can also be used to prepare compoundshaving other different substituents either on the naphthalene ring or onthe propionic acid side chain, as shown in the following structure, 6,where R can be any substituent that does not interfere with the 10reactions. Examples of R include, but are not limited to, hydrogen,alkyl, alkoxy, alkylthio, alkoxyalkyl, alkylthioalkyl, dialkylamino,halogen, aryl, aryloxy, arylthio, alkanesulfonyl, alkanesulfinyl,silyloxy, protected ketone, and aldehyde (e.g., ketal and acetal).

[0276] The major starting materials for this synthesis would have thefollowing structures, e.g., 7 and 8, in which X is a halogen atom, forexample Cl, Br, or I. Compound 8 is a derivative of acrylic acid, inwhich Y is a heteroatom, preferably oxygen or nitrogen.

[0277] The experiments described below were carried out in etherealsolution starting from 2-bromo-6-methoxynaphthalene at pressures rangingfrom 0.1 to 100 atmospheres. Other suitable solvents include, forexample, ethers, alkanes, and aromatic hydrocarbons. The temperature canrange from-100° C. to ±150° C. Metals that can be used for thesereactiond include magnesium, lithium, sodium, potassium, calcium,palladium, copper, and aluminum. This reaction can also be catalyzed bycopper (I) halides alone, or in the presence of other co-calalysts, suchas phosphines and boron trifluoride. Chiral auxiliary groups used toinduce asymmetric Michael addition include those derived from L- orD-menthol, L- or D-camphor, proline-derived amines and amides. Thereaction can also be carried out in the presence of other asymmetriccompounds, such as (−)-sparteine, which can induce asymmetric Michaeladditions under similar reaction conditions.

[0278] The starting materials for this synthesis can have the structuresillustrated by 7 and 8. The methyl group for the methylation can bederived from methyl iodide, dimethyl sulphate, methyl arenesulfonate,methyl alkanesulfonate, etc.

Example 6 Preparation of L-menthyl trans-2-methyl-2-pentenoate

[0279] To a 100-mL round-bottomed flask trans-2-methyl-2-pentenoic acid(11.4 g, 100 mmol) and thionyl chloride (18 mL, 210 mmol) were added.Bubbles evolved from the light-yellow solution immediately. The mixturewas stirred at room temperature for 5 min and then heated to reflux for30 min, during which time the mixture turned brown. Unreacted thionylchloride was removed by distillation. L-menthol (15.4 g, 99 mmol) wasadded to the formed acyl chloride and the mixture was heated in a 160°C. oil bath of for 1 hour, at which time the evolution of HCl ceased.The mixture was transferred into a separatory funnel and the flaskrinsed with hexanes (100 mL). The hexanes solution was then washed withaqueous NaHCO₃ solution and water. Removal of hexanes in vacuo followedby vacuum distillation gave a light yellow oil, 18.7 g; yield: 75%.

Example 7 Preparation of L-menthyl3-[2-(6-Methoxynaphthyl)]-2,2-dimethylpentanoate

[0280] To a 25-mL, three-necked round-bottomed flask equipped with astir bar and a condenser, freshly ground magnesium turnings (0.29 g,12.1 mmol) were added. The condenser and the flask were sealed withrubber septa and 5 mL of freshly distilled dry THF was injected. Argonwas bubbled into the reaction flask to replace air, followed by dropwiseinjection of 1,2-dibromoethane (0.2 mL, 2.3 mmol). The reaction mixturestarted to reflux shortly without external heating. A solution of2-bromo-6-methoxynaphthalene (Aldrich, 2.37 g, 10 mmol) in dry THF wassyringed dropwise into the flask at a speed to maintain the reflux.After all the solution was added, the mixture was heated to maintainreflux for 40 min before being cooled in an ice-water bath. L-menthyltrans-2-methyl-2-pentenoate (2.2 g, 8.7 mmol) was dissolved in 5 mL ofdry THF and the solution was injected into the flask. The ice-water bathwas removed and the mixture was stirred at room temperature for 1.5 hbefore methyl iodide (0.62 mL, 10 mmol) was added via a syringe. Thereaction proceeded for 15 min before being quenched with water. Productwas extracted with ether and the ethereal solution was dried overmagnesium sulfate. Removal of ether in vacuo gave a light yellow oil,3.33 g. Column chromatography (silica gel, hexanes:ethyl acetate=50:1)provided a yellow oil, 2.85 g, whose ¹H NMR spectrum showed the presenceof L-menthyl 3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoate as themajor product. Yield by NMR: 77%. The two diastereomers exist in equalamount.

Example 8 Preparation of (−)-8-phenylmethyl trans-2-methyl-2-pentenoate

[0281] To a 50-mL round-bottomed flask trans-2-methyl-2-pentenoic acid(2.2 g, 19.3 mmol) and thionyl chloride (5 mL, 58 mmol) were added.Bubbles evolved from the light yellow solution immediately. The mixturewas stirred at room temperature for 5 min and then heated at reflux for30 min. Unreacted thionyl chloride was removed by heating the mixture inan oil bath at 160° C. (−)-8-Phenylmenthol (Aldrich, 0.95 g, 4.1 mmol)was added to the acyl chloride and the mixture was heated in an oil bathat 190° C. for 30 min. Dilute aqueous KOH solution was added into themixture and the product was extracted with ether. Evaporation of etherprovided a light brown oil which is further purified by columnchromatography (silica gel, hexanes:ether=50:1) to give a light yellowoil, 1.28 g; yield: 94.8%. ¹H NMR showed that it was the desiredproduct, but contained some cis isomer, the trans:cis ratio being 5:1.

Example 9 Preparation of (−)-8-phenylmethyl3-[2-(6-Methoxynaphthyl)]-2.2-dimethyl-pentanoate

[0282] To a 25-mL, three-necked round-bottomed flask equipped with astir bar and a condenser, freshly ground magnesium turnings (0.171 g,7.1 mmol) were added. The condenser and the flask were sealed withrubber septa and 5 mL of freshly distilled dry THF was injected. Argonwas bubbled into the reaction flask to replace air, followed by dropwiseinjection of 1,2-dibromoethane (0.18 mL, 2.1 mmol). The reaction mixturestarted to reflux shortly without external heating. A solution of2-bromo-6-methoxynaphthalene (1.20 g, 5.1 mmol) in dry THF (10 mL) wassyringed dropwise into the flask at a speed to maintain the reflux.After all the solution was added, the mixture was heated to maintainreflux for 45 min before being cooled in an ice-water bath.(−)-8-Phenylmenthyl trans-2-methyl-2-pentenoate (1.27 g, 3.8 mmol) wasdissolved in 5 mL of dry THF and the solution was injected into theflask. The ice-water bath was removed and the mixture was stirred atroom temperature for 1.5 h before methyl iodide (0.4 mL, 6.4 mmol) wassyringed into the flask. After 15 min the reaction was quenched withaqueous NH₄Cl solution. The product was extracted with ether, theethereal solution was dried over magnesium sulfate, and the ether wasremoved in vacuo to yield a light-yellow thick oil. Columnchromatography (silica gel, hexanes:ethyl acetate=50:1˜20:1) provided ayellow oil, 1.04 g, whose ¹H NMR spectrum showed that the twodiastereomers exist in a ratio of about. 1.7:1. Yield: 77% based onreacted starting material.

[0283] The present invention thus provides direct, one-pot methods forthe asymmetric synthesis of esters of (+)- and(−)-3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid and esters ofother substituted 3-(2-naphthyl)propionic acids. These esters can beeasily hydrolyzed into their corresponding free acids.

[0284] The invention being thus described, it will be obvious that thesame can be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the present invention, andall such modifications and equivalents as would be obvious to oneskilled in the art are intended to be included within the scope of thefollowing claims.

[0285] The contents of each of the references cited herein are herebyincorporated by reference in their entirety.

What is claimed is:
 1. A method for repressing weight gain or reducingweight in a male patient, comprising administering(+)-Z-bisdehydrodoisynolic acid in a dosage effective to repress weightgain or reduce weight to a male patient suffering from, or disposed to,weight gain.
 2. The method of claim 1, wherein said dosage is in therange of from about 0.1 μg/kg/day to about 100 mg/kg/day.
 3. A methodfor treating or preventing a disease, condition, or symptom selectedfrom the group consisting of prostatic disease, peri- or post-menopausalsymptoms, an estrogen-responsive condition that no longer responds totreatment with conventional steroidal estrogens, an estrogen-responsiveuterine cancer, breast cancer, ovarian follicle atresia, a disease orcondition caused or prolonged by free radicals, cardiovascular disease,hyperlipidemia, hypercholesterolemia, hyperglycemia, Alzheimer's diseaseand pattern baldness, comprising administering an estrogenic carboxylicacid in a dosage effective to treat or prevent said disease, symptom, orcondition to a patient suffering from, or disposed to, said disease,symptom, or condition.
 4. The method of claim 3, wherein said estrogeniccarboxylic acid is selected from the group consisting of a doisynolicacid, an allenolic acid, a phenylcyclohexenecarboxylic acid, ahydroxyphenylcyclohexenecarboxylic acid, a phenylcyclohexanecarboxylicacid, a hydroxyphenylcyclohexanecarboxylic acid, ahydroxytetrahydroanthracenecarboxylic acid, and atetrahyroanthracenecarboxylic acid.
 5. The method of claim 4, whereinsaid estrogenic carboxylic acid is selected from the group consisting of(+)-doisynolic acid, (−)-Z-bisdehydro-doisynolic acid,(+)-Z-bisdehydrodoisynolic acid, (±)-Z-bisdehydrodoisynolic acid,(−)-allenolic acid, (+)-allenolic acid,1-(p-hydroxyphenyl)-6-ethyl-5-methylcyclohexene-4-carboxylic acid,1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohexene-4-carboxylic acid,1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohexene-4-carboxylicacid, 4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexanecarboxylic acid,1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2-carboxylicacid, 1-phenyl-2-ethyl-3-methylcyclohexene-4-carboxylic acid, and1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid, or apharmaceutically acceptable salt, ester, or anhydride thereof.
 6. Themethod of claim 5, wherein said estrogenic carboxylic acid is selectedfrom the group consisting of (−)-Z-bisdehydrodoisynolic acid,(+)-Z-bisdehydrodoisynolic acid, and (±)-Z-bisdehydrodoisynolic acid. 7.The method of claim 3, wherein said dosage is in the range of from about0.1 μg/kg/day to about 100 mg/kg/day.
 8. A method for treating orpreventing osteoporosis comprising administering an estrogeniccarboxylic acid selected from the group consisting of a doisynolic acid,a phenylcyclohexenecarboxylic acid, a hydroxyphenylcyclohexenecarboxylicacid, a phenylcyclohexanecarboxylic acid, ahydroxyphenylcyclohexanecarboxylic acid, ahydroxytetrahydroanthracenecarboxylic acid, and atetrahydroanthracenecarboxylic acid in a dosage effective to treat orprevent osteoporosis to a male or female patient suffering from, ordisposed to, osteoporosis.
 9. The method of claim 8 wherein saidestrogenic carboxylic acid is selected from the group consisting of(+)-doisynolic acid, (−)-Z-bisdehydro-doisynolic acid,(+)-Z-bisdehydrodoisynolic acid, (±)-Z-bisdehydrodoisynolic acid,1-(p-hydroxyphenyl)-6-ethyl-5-methylcyclohexene-4-carboxylic acid,1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohexene-4-carboxylic acid,1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohexene-4-carboxylicacid, 4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexanecarboxylic acid,1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2-carboxylicacid, 1-phenyl-2-ethyl-3-methylcyclohexene-4-carboxylic acid, and1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid, or apharmaceutically acceptable salt, ester, or anhydride thereof.
 10. Themethod of claim 9, wherein said estrogenic carboxylic acid is selectedfrom the group consisting of (−)-Z-bisdehydrodoisynolic acid,(+)-Z-bisdehydrodoisynolic acid, and (±)-Z-bisdehydrodoisynolic acid.11. The method of claim 8, wherein said dosage is in the range of fromabout 0.1 μg/kg/day to about 100 mg/kg/day.